The present invention relates to a new plant breeding process. The process improves the agronomic performance of crop plants by using genetic material that is also used in classical breeding. Instead of sexually recombining entire genomes at random, as is done in classical breeding, specific genetic elements are rearranged in vitro and inserted back into individual plant cells. plants obtained through this new plant breeding process do not contain foreign nucleic acid but only contain nucleic acid from the plant species selected for transformation or plants that are sexually compatible with the selected plant species. plants developed through this new plant breeding process are provided. In particular, potato plants displaying improved tuber storage and health characteristics are provided.
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1. A method of modifying a trait of a selected plant by stably integrating only a desired polynucleotide into the genome of said plant, comprising (a) transforming said plant with said desired polynucleotide, wherein said desired polynucleotide consists essentially of (i) a nucleic acid sequence that is native to the selected plant, native to a plant from the same species as the selected plant, or native to a plant that is sexually interfertile with said selected plant, wherein (ii) the desired polynucleotide does not contain foreign DNA that is not from the selected plant species or a plant that is sexually compatible with the selected plant species and (iii) the desired polynucleotide does not comprise a nucleotide sequence that is identical in nucleotide sequence to an agrobacterium t-DNA border sequence; (b) selecting plant cells comprising only said desired polynucleotide in the genome; and (c) obtaining a stably transformed plant from the selected transformed plant cells, wherein the transformed plant contains only the desired polynucleotide stably integrated into its genome and wherein the stably transformed plant exhibits a trait that is modified in comparison to the selected plant.
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This application is a continuation-in-part of U.S. application Ser. No. 10/369,324 filed on Feb. 20, 2003, and claims priority to U.S. provisional application Ser. Nos. 60/357,661 filed on Feb. 20, 2002 and 60/377,602 filed on May 6, 2002, which applications are all incorporated herein by reference.
Carrier DNA: a “carrier DNA” is a DNA segment that is used to carry certain genetic elements and deliver them into a plant cell. In conventional foreign DNA transfer, this carrier DNA is often the T-DNA of Agrobacterium, delineated by border sequences. The carrier DNA described here is obtained from the selected plant species to be modified and contains ends that may be structurally and functionally different from T-DNA borders but shares with such T-DNAs the ability to support both DNA transfer from Agrobacterium to the nuclei of plant cells or certain other eukaryotes and the subsequent integration of this DNA into the genomes of such eukaryotes.
Consisting essentially of: a composition “consisting essentially of” certain elements is limited to the inclusion of those elements, as well as to those elements that do not materially affect the basic and novel characteristics of the inventive composition. Thus, so long as the composition does not affect the basic and novel characteristics of the instant invention, that is, does not contain foreign DNA that is not from the selected plant species or a plant that is sexually compatible with the selected plant species, then that composition may be considered a component of an inventive composition that is characterized by “consisting essentially of” language.
Degenerate primer: a “degenerate primer” is an oligonucleotide that contains sufficient nucleotide variations that it can accommodate base mismatches when hybridized to sequences of similar, but not exact, homology.
Dicotyledon (dicot): a flowering plant whose embryos have two seed leaves or cotyledons. Examples of dicots include, but are not limited to, tobacco, tomato, potato, sweet potato, cassaya, legumes including alfalfa and soybean, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, and cactus.
Regulatory sequences: refers to those sequences which are standard and known to those in the art, that may be included in the expression vectors to increase and/or maximize transcription of a gene of interest or translation of the resulting RNA in a plant system. These include, but are not limited to, promoters, peptide export signal sequences, introns, polyadenylation, and transcription termination sites. Methods of modifying nucleic acid constructs to increase expression levels in plants are also generally known in the art (see, e.g. Rogers et al., 260 J. Biol. Chem. 3731-38, 1985; Cornejo et al., 23 Plant Mol. Biol. 567: 81,1993). In engineering a plant system to affect the rate of transcription of a protein, various factors known in the art, including regulatory sequences such as positively or negatively acting sequences, enhancers and silencers, as well as chromatin structure may have an impact. The present invention provides that at least one of these factors may be utilized in engineering plants to express a protein of interest. The regulatory sequences of the present invention are native genetic elements, i.e., are isolated from the selected plant species to be modified.
Foreign: “foreign,” with respect to a nucleic acid, means that that nucleic acid is derived from non-plant organisms, or derived from a plant that is not the same species as the plant to be transformed or is not derived from a plant that is not interfertile with the plant to be transformed, does not belong to the species of the target plant. According to the present invention, foreign DNA or RNA represents nucleic acids that are naturally occurring in the genetic makeup of fungi, bacteria, viruses, mammals, fish or birds, but are not naturally occurring in the plant that is to be transformed. Thus, a foreign nucleic acid is one that encodes, for instance, a polypeptide that is not naturally produced by the transformed plant. A foreign nucleic acid does not have to encode a protein product. According to the present invention, a desired transgenic plant is one that does not contain any foreign nucleic acids integrated into its genome.
Native genetic elements, on the other hand, can be incorporated and integrated into a selected plant species genome according to the present invention. Native genetic elements are isolated from plants that belong to the selected plant species or from plants that are sexually compatible with the selected plant species. For instance, native DNA incorporated into cultivated potato (Solanum tuberosum) can be derived from any genotype of S. tuberosum or any genotype of a wild potato species that is sexually compatible with S. tuberosum (e.g., S. demissum).
Gene: “gene” refers to the coding region and does not include nucleotide sequences that are 5′- or 3′- to that region. A functional gene is the coding region operably linked to a promoter or terminator.
Genetic rearrangement: refers to the reassociation of genetic elements that can occur spontaneously in vivo as well as in vitro which introduce a new organization of genetic material. For instance, the splicing together of polynucleotides at different chromosomal loci, can occur spontaneously in vivo during both plant development and sexual recombination. Accordingly, recombination of genetic elements by non-natural genetic modification techniques in vitro is akin to recombination events that also can occur through sexual recombination in vivo.
In frame: nucleotide triplets (codons) are translated into a nascent amino acid sequence of the desired recombinant protein in a plant cell. Specifically, the present invention contemplates a first nucleic acid linked in reading frame to a second nucleic acid, wherein the first nucleotide sequence is a gene and the second nucleotide is a promoter or similar regulatory element.
Integrate: refers to the insertion of a nucleic acid sequence from a selected plant species, or from a plant that is from the same species as the selected plant, or from a plant that is sexually compatible with the selected plant species, into the genome of a cell of a selected plant species. “Integration” refers to the incorporation of only native genetic elements into a plant cell genome. In order to integrate a native genetic element, such as by homologous recombination, the present invention may “use” non-native DNA as a step in such a process. Thus, the present invention distinguishes between the “use of” a particular DNA molecule and the “integration” of a particular DNA molecule into a plant cell genome.
Introduction: as used herein, refers to the insertion of a nucleic acid sequence into a cell, by methods including infection, transfection, transformation or transduction.
Isolated: “isolated” refers to any nucleic acid or compound that is physically separated from its normal, native environment. The isolated material may be maintained in a suitable solution containing, for instance, a solvent, a buffer, an ion, or other component, and may be in purified, or unpurified, form.
Leader: Transcribed but not translated sequence preceding (or 5′ to) a gene.
LifeSupport Vector: a LifeSupport vector is a construct that contains an expressable selectable marker gene, such as a neomycin phosphotransferase marker, that is positioned between T-DNA or T-DNA-like borders. The LifeSupport vector may be modified to limit integration of such a marker, as well as other polynucleotides, that are situated between the border or border-like sequences, into a plant genome. For instance, a LifeSupport vector may comprise a mutated virD2, codA::upp fusion, or any combination of such genetic elements. Thus, a modified virD2 protein will still support T-DNA transfer to plant nuclei but will limit the efficiency of a subsequent genomic integration of T-DNAs (Shurvinton et al., Proc Natl Acad Sci USA, 89: 11837-11841, 1992; Mysore et al., Mol Plant Microbe Interact, 11: 668-683, 1998). Alternatively, codA::upp gene fusion can be used as negative selectable marker prior to regeneration. In one preferred construct, the LifeSupport vector comprises the npt marker operably linked to the yeast ADH terminator element.
Monocotyledon (monocot): a flowering plant whose embryos have one cotyledon or seed leaf. Examples of monocots include, but are not limited to turf grass, maize, rice, oat, wheat, barley, sorghum, orchid, iris, lily, onion, and palm.
Native: a “native” genetic element refers to a nucleic acid that naturally exists in, orginates from, or belongs to the genome of a plant that is to be transformed. Thus, any nucleic acid, gene, polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is isolated either from the genome of a plant or plant species that is to be transformed or is isolated from a plant or species that is sexually compatible or interfertile with the plant species that is to be transformed, is “native” to, i.e., indigenous to, the plant species. In other words, a native genetic element represents all genetic material that is accessible to plant breeders for the improvement of plants through classical plant breeding. Any variants of a native nucleic acid also are considered “native” in accordance with the present invention. In this respect, a “native” nucleic acid may also be isolated from a plant or sexually compatible species thereof and modified or mutated so that the resultant variant is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or 60% similar in nucleotide sequence to the unmodified, native nucleic acid isolated from a plant. A native nucleic acid variant may also be less than about 60%, less than about 55%, or less than about 50% similar in nucleotide sequence.
A “native” nucleic acid isolated from a plant may also encode a variant of the naturally occurring protein product transcribed and translated from that nucleic acid. Thus, a native nucleic acid may encode a protein that is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or 60% similar in amino acid sequence to the unmodified, native protein expressed in the plant from which the nucleic acid was isolated.
Naturally occurring nucleic acid: this phrase means that the nucleic acid is found within the genome of a selected plant species and may be a DNA molecule or an RNA molecule. The sequence of a restriction site that is normally present in the genome of a plant species can be engineered into an exogenous DNA molecule, such as a vector or oligonucleotide, even though that restriction site was not physically isolated from that genome. Thus, the present invention permits the synthetic creation of a nucleotide sequence, such as a restriction enzyme recognition sequence, so long as that sequence is naturally occurring in the genome of the selected plant species or in a plant that is sexually compatible with the selected plant species that is to be transformed.
Operably linked: combining two or more molecules in such a fashion that in combination they function properly in a plant cell. For instance, a promoter is operably linked to a structural gene when the promoter controls transcription of the structural gene.
P-DNA: according to the present invention, P-DNA (“plant-DNA”) is isolated from a plant genome and comprises at each end, or at only one end, a T-DNA border-like sequence. The border-like sequence preferably shares at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or at least 95%, but less than 100% sequence identity, with a T-DNA border sequence from an Agrobacterium species, such as Agrobacterium tumefaciens or Agrobacterium rhizogenes. Thus, P-DNAs can be used instead of T-DNAs to transfer a nucleotide sequence from Agrobacterium to another polynucleotide sequence. The P-DNA may be modified to facilitate cloning and should preferably not naturally encode proteins or parts of proteins. The P-DNA is characterized in that it contains, at each end, at least one border sequence, referred to as either a “P-DNA border sequence” or “P-DNA border-like sequence,” which are interexchangeable terms. See the definition of a “border sequence” and “border-like” above. A P-DNA may also be regarded as a “T-DNA-like” sequence, see definition below.
Plant: includes angiosperms and gymnosperms such as potato, tomato, tobacco, alfalfa, lettuce, carrot, strawberry, sugarbeet, cassaya, sweet potato, soybean, maize, turf grass, wheat, rice, barley, sorghum, oat, oak, eucalyptus, walnut, and palm. Thus, a plant may be a monocot or a dicot. The word “plant,” as used herein, also encompasses plant cells, seed, plant progeny, propagule whether generated sexually or asexually, and descendents of any of these, such as cuttings or seed. Plant cells include suspension cultures, callus, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, seeds and microspores. Plants may be at various stages of maturity and may be grown in liquid or solid culture, or in soil or suitable media in pots, greenhouses or fields. Expression of an introduced leader, trailer or gene sequences in plants may be transient or permanent. A “selected plant species” may be, but is not limited to, a species of any one of these “plants.”
Precise breeding: refers to the improvement of plants by stable introduction of nucleic acids, such as native genes and regulatory elements isolated from the selected plant species, or from another plant in the same species as the selected plant, or from species that are sexually compatible with the selected plant species, into individual plant cells, and subsequent regeneration of these genetically modified plant cells into whole plants. Since no unknown or foreign nucleic acid is permanently incorporated into the plant genome, the inventive technology makes use of the same genetic material that is also accessible through conventional plant breeding.
Plant species: the group of plants belonging to various officially named plant species that display at least some sexual compatibility.
Plant transformation and cell culture: broadly refers to the process by which plant cells are genetically modified and transferred to an appropriate plant culture medium for maintenance, further growth, and/or further development.
Recombinant: as used herein, broadly describes various technologies whereby genes can be cloned, DNA can be sequenced, and protein products can be produced. As used herein, the term also describes proteins that have been produced following the transfer of genes into the cells of plant host systems.
Selectable marker: a “selectable marker” is typically a gene that codes for a protein that confers some kind of resistance to an antibiotic, herbicide or toxic compound, and is used to identify transformation events. Examples of selectable markers include the streptomycin phosphotransferase (spt) gene encoding streptomycin resistance, the phosphomannose isomerase (pmi) gene that converts mannose-6-phosphate into fructose-6 phosphate; the neomycin phosphotransferase (nptII) gene encoding kanamycin and geneticin resistance, the hygromycin phosphotransferase (hpt or aphiv) gene encoding resistance to hygromycin, acetolactate synthase (als) genes encoding resistance to sulfonylurea-type herbicides, genes coding for resistance to herbicides which act to inhibit the action of glutamine synthase such as phosphinothricin or basta (e.g., the bar gene), or other similar genes known in the art.
Sense suppression: reduction in expression of an endogenous gene by expression of one or more an additional copies of all or part of that gene in transgenic plants.
T-DNA-Like: a “T-DNA-like” sequence is a nucleic acid that is isolated from a selected plant species, or from a plant that is sexually compatible with the selected plant species, and which shares at least 75%, 80%, 85%, 90%, or 95%, but not 100%, sequence identity with Agrobacterium species T-DNA. The T-DNA-like sequence may contain one or more border or border-like sequences that are each capable of integrating a nucleotide sequence into another polynucleotide. A “P-DNA,” as used herein, is an example of a T-DNA-like sequence.
Trailer: Transcribed but not translated sequence following (or 3′to) a gene.
Transcribed DNA: DNA comprising both a gene and the untranslated leader and trailer sequence that are associated with that gene, which is transcribed as a single mRNA by the action of the preceding promoter.
Transcription and translation terminators: the expression vectors of the present invention typically have a transcription termination region at the opposite end from the transcription initiation regulatory region. The transcription termination region may be selected, for stability of the mRNA to enhance expression and/or for the addition of polyadenylation tails added to the gene transcription product (Alber & Kawasaki, Mol. & Appl. Genetics 4: 19-34, 1982). Illustrative transcription termination regions include the E9 sequence of the pea RBCS gene (Mogen et al., Mol. Cell Biol., 12: 5406-14, 1992) and the termination signals of various ubiquitin genes.
Transformation of plant cells: a process by which DNA is stably integrated into the genome of a plant cell. “Stably” refers to the permanent, or non-transient retention and/or expression of a polynucleotide in and by a cell genome. Thus, a stably integrated polynucleotide is one that is a fixture within a transformed cell genome and can be replicated and propagated through successive progeny of the cell or resultant transformed plant. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of nucleic acid sequences into a prokaryotic or eukaryotic host cell, including Agrobacterium-mediated transformation protocols, viral infection, whiskers, electroporation, heat shock, lipofection, polyethylene glycol treatment, micro-injection, and particle bombardment.
Transgene: a gene that will be inserted into a host genome, comprising a protein coding region. In the context of the instant invention, the elements comprising the transgene are isolated from the host genome.
Transgenic plant: a genetically modified plant which contains at least one transgene.
Using/Use of: The present invention envisions the use of nucleic acid from species other than that of the selected plant species to be transformed to facilitate the integration of native genetic elements into a selected plant genome, so long as such foreign nucleic acid is not stably integrated into the same host plant genome. For instance, the plasmid, vector or cloning construct into which native genetic elements are cloned, positioned or manipulated may be derived from a species different to that from which the native genetic elements were derived.
Variant: a “variant,” as used herein, is understood to mean a nucleotide or amino acid sequence that deviates from the standard, or given, nucleotide or amino acid sequence of a particular gene or protein. The terms, “isoform,” “isotype,” and “analog” also refer to “variant” forms of a nucleotide or an amino acid sequence. An amino acid sequence that is altered by the addition, removal or substitution of one or more amino acids, or a change in nucleotide sequence, may be considered a “variant” sequence. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. A variant may have “non-conservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted may be found using computer programs well known in the art such as Vector NTI Suite (InforMax, MD) software.
It is understood that the present invention is not limited to the particular methodology, protocols, vectors, and reagents, etc., described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a gene” is a reference to one or more genes and includes equivalents thereof known to those skilled in the art and so forth. Indeed, one skilled in the art can use the methods described herein to express any native gene (known presently or subsequently) in plant host systems.
P-DNA Vectors
Agrobacterium-mediated transformation methods are the preferred means of incorporating recombined DNA into plant cells. According to the present invention, a binary vector was developed to produce genetically modified potato plants that contain only native potato nucleic acids. Such a vector is different from conventional, Agrobacterium-mediated transformation vectors in three ways: (1) instead of an Agrobacterium-derived T-DNA sequence delineated by T-DNA borders, the present vector contains a native plant DNA (P-DNA) fragment that is flanked by border-like sequences, which support P-DNA transfer from Agrobacterium to plant cells although they are structurally and functionally different from T-DNA borders, (2) the backbone of the present vector may contain a marker that, if integrated into the plant cell's genome, prevents these cells from developing into mature plants, and (3) the present vector does not contain a foreign selectable marker gene between P-DNA termini.
The present invention demonstrates, surprisingly, that P-DNA fragments flanked by border-like sequences support DNA transfer from Agrobacterium into plant cells. P-DNA can be isolated from the genome of any plant by using primers that are designed on the basis of homology between the termini of a potato P-DNA and conventional T-DNA borders. Such fragments can then be tested and, if efficacious, used to transform that plant with native DNA exclusively. It is also possible to search plant genomic databases for DNA fragments with regions that show homology with T-DNA borders by using programs such as ‘blastn’ (Altschul et al., J Mol Biol 215: 403-10, 1990). The identified P-DNAs may then be modified to increase their utility. For instance, internal fragments of the isolated P-DNAs may be deleted and restriction sites may be added to facilitate cloning. It may also be efficacious to introduce point mutations at the terminal sequences to render the P-DNA more effective in transferring DNA.
Any gene expression cassette can be inserted between P-DNA border-like sequences. For potato transformations, such an expression cassette could consist of a potato promoter, operably linked to a potato gene and/or a leader or trailer sequence associated with that gene, and followed by a potato terminator. The expression cassette may contain additional potato genetic elements such as a signal peptide sequence fused in frame to the 5′-end of the gene, and a potato intron that could, for instance, be placed between promoter and gene-of-interest to enhance expression. For transformation of wheat with a modified P-DNA, all genetic elements that are inserted on the wheat P-DNA, including the P-DNA itself would be derived from wheat or plant species that are sexually compatible with wheat.
Another way to isolate P-DNAs is by generating a library of Agrobacterium strains that contain random plant DNA fragments instead of a T-DNA flanking a selectable marker gene. Explants infected with this library can be placed on proliferation medium that contains an appropriate selectable agent to identify P-DNAs that support the transfer of the marker gene from the vector in Agrobacterium to the plant cell.
It is possible that not just the native modified P-DNA, but also additional plasmid sequences are co-transferred from Agrobacterium to the plant cell during the transformation process. For the purposes of the present invention, this is an undesirable process because such plasmid “backbone” sequences represent non-plant, foreign DNA, such as bacterial DNA. The present invention prevents transformed plant cells that contain backbone sequences from developing into mature plants. Thus, the present invention makes it possible to distinguish backbone-containing and backbone-free transformation events during the regenerated shoot phase.
The method to select or screen against backbone integration events relies on the presence of an expression cassette for a marker, such as the isopentenyl phosphotransferase (IPT) gene, in the vector backbone, outside of the P-DNA. Upon backbone integration, the accumulation of IPT-induced cytokinin will alter the shape of transformed shoots, and prevent these shoots to develop roots. Instead of the IPT gene, any other gene that alters the shape, texture or color of the transformed plant's leaves, roots, stem, height or some other morphological feature can be used to screen and/or select against backbone integration events. Such a gene is referred to herein as a “backbone integration marker.” Thus, the transformed plant that exhibits an altered morphological feature attributable to the expression of the backbone integration marker gene is known to, contain in its genome foreign DNA in addition to the desired P-DNA. Accordingly, plants that exhibit a phenotype associated with the backbone integration marker are not desired.
The present invention is not limited to the use of only an IPT gene as a backbone integration marker; other genes can be used in such fashion. For example, a backbone integration marker may be an Agrobacterium transzeatine synthase (TZS) gene (Krall et al., FEBS Lett 527: 315-8, 2002) or a recessive Arabidopsis gene hod (Catterou et al., Plant J 30: 273-87, 2002). This method can be more easily applied for use in the present invention than some methods that insert toxic genes in vector backbone sequences. See, for instance, EP 1 009,842.
By positioning a backbone integration marker gene, such as a functional cytokinin gene upstream or downstream of the P-DNA, it is straightforward to distinguish between transformation events. Transformed plants that exhibit an altered morphological feature are discarded because they contain non-native DNA sequences integrated into the genome.
Another strategy for identifying plants that are stably transformed with only native DNA, is to employ the polymerase chain reaction. By using primers that are specifically designed to detect backbone sequences, plants can be identified and discarded that contain foreign backbone sequences in addition to the P-DNA. Other primer sets can subsequently be used to confirm the intact transfer of the P-DNA. Thus, by either using the expression of a gene to change a morphological feature of a plant, or by screening for stably integrated foreign DNA in a transformed plant, plants stably transformed with only native DNA sequences can be identified and selected.
Genetic elements from a particular host plant can be inserted into the P-DNA sequence of a binary vector capable of replication in both E. coli and Agrobacterium. Introduction of the resulting vectors into disarmed Agrobacterium strains such as LBA4404 can be accomplished through electroporation, triparental mating or heat-shock treatment of chemically competent cells. The new strains can then be used to transform individual plant cells through infection of whole plants or explants.
Genetic elements from a particular host plant can be inserted into the P-DNA sequence of a binary vector capable of replication in both E. coli and Agrobacterium. Introduction of the resulting vectors into Agrobacterium strains such as LBA4404 can be accomplished through electroporation, triparental mating or heat-shock treatment of chemically competent cells. The new strains can then be used to transform individual plant cells through infection of whole plants or explants. LBA4404 contains the disarmed Ti-plasmid pAL4404, which carries the virulence functions and a streptomycin resistance gene.
LifeSupport Vectors
Although the stable integration of bacterial marker genes into the genomes of plant cells facilitates the identification of transformation events, such modifications of plant genomes are undesirable because marker genes represent foreign DNA. Use of a foreign marker gene can be avoided by developing new Agrobacterium-based transformation methods.
One preferred embodiment is a novel method that relies on the use of two Agrobacterium strains: one strain containing a binary vector with a selectable marker gene intended for transient expression in plant nuclei, and another strain carrying the P-DNA with the actual sequences of interest intended for stable integration in plant genome (see Example 7).
Upon co-infection with the Agrobacterium strains, some plant cells will receive both a T-DNA with the marker gene and a P-DNA with the sequences of interest. Instead of subsequently selecting for stable integration of the marker gene by subjecting the infected explants for a long period of time to the appropriate antibiotic, explants are only briefly exposed to the antibiotic. In this way, all plant cells that transiently express the marker gene will survive. Because T-DNAs will in most cases degrade due to endogenous nuclease activities rather than stably integrate into their host's genome, the majority of plant cells that survived the transient selection are shown here to develop into shoots lacking a marker gene. The present invention, furthermore, demonstrates that a significant proportion of these marker-free shoots contain stably integrated P-DNAs.
There are various tools to enhance the efficiency of marker-free transformation. First, the present invention demonstrates that this frequency can be increased by sequentially infecting explants with two Agrobacterium strains carrying the T-DNA/marker and P-DNA/sequences-of-interest, respectively. Explants are first infected with the P-DNA strain, and after about 4 to 6 hours with the T-DNA strain.
Second, the T-DNA strain can be modified to express an omega-mutated virD2 gene. The modified virD2 protein will still support T-DNA transfer to plant nuclei but limit the efficiency of a subsequent genomic integration of T-DNAs (Shurvinton et al., Proc Natl Acad Sci USA, 89: 11837-11841, 1992; Mysore et al., Mol Plant Microbe Interact, 11: 668-683, 1998). The most preferred method of expressing a modified virD2 gene is by inserting an omega-mutated virD2 gene driven by the virD promoter in the backbone of the T-DNA vector.
Third, stable T-DNA integration can be further impaired by inserting telomere sequences close to the left- and right-border sequences of the T-DNA (Chiurazzi & Signer, Plant Mol. Biol., 26: 923-934, 1994).
Fourth, the size of the T-DNA region carrying the marker gene can be increased to enhance the frequency of T-DNAs and P-DNAs moving together into the plant cell nucleus, and to reduce the frequency of genomic integration of the T-DNA.
Fifth, the frequency of T-DNAs and P-DNAs moving together into the plant cell nucleus can also be enhanced by using a single Agrobacterium strain carrying two compatible binary vectors with the T-DNA and P-DNA, respectively. An example of two compatible binary vectors are a pSIM 1301-derived vector and a pBI121-derived vector.
Because the transiently expressed marker gene will usually not integrate into the plant genome, it is not necessary that both this gene and its regulatory sequences represent native DNA. In fact, it may be advantageous to use foreign regulatory sequences to promote high levels of transient gene expression in infected plant cells. A surprising discovery of the present invention is that an expression cassette containing the GUS gene followed by the terminator of the yeast alcohol dehydrogenase 1 (ADH1) was transiently expressed at high levels in potato cells. A similar construct with the yeast CYC1 terminator, however, did not function adequately. It may also be possible to enhance transient expression levels by operably linking a marker gene to a non-native promoter. Examples of such promoters are, e.g., synthetic promoters such as glucocorticoid-inducible promoters (Mori et al., Plant J., 27: 79-86, 2001; Bohner et al., Mol. Gen. Genet., 264: 860-70 2001), and non-native promoters such as the 35S promoters of cauliflower mosaic virus and figwort mosaic virus, and fungal promoters.
As an alternative to the two-strain Agrobacterium-mediated transformation approach described above, plants may also be transformed with a single strain that contains a P-DNA with both a native marker gene and the actual sequences of interest. The present invention demonstrates that it is possible to use salt tolerance genes as native markers for transformation. Such salt tolerance genes include crop homologs of the Arabidopsis genes SOS1 (Shi et al., Nat Biotechnol. 2002), AtNHX1 (Apse et al., Science. 285: 1256-8, 1999), Avp1 (Gaxiola et al., Proc Natl Acad Sci USA. 98: 11444-9, 2001), and CBF3 (Kasuga et al., Nat Biotechnol. 17: 287-91, 1999).
The rearrangements of genetic elements accomplished through the inventive Precise Breeding methodology could also occur spontaneously through the process of genetic recombination. For instance, all plants contain elements that can transpose from one to another chromosomal location. By inserting into promoters or genes, such transposable elements can enhance, alter, and/or reduce gene expression. For instance, the AMu4 insertion of the maize Mutator element in the promoter of the transcriptional regulator gene P-wr causes stripy red pericarps. Insertion of the same element in the promoter of the leaf-specific MADS-box gene ZMM19 resulted in expression of this gene in the inflorescences of maize, causing a foliaceous elongation of the glumes and other changes in male and female inflorescences, resulting in the famous phenotype of pod corn. Because of its bizarre tassels and ears, pod corn was of religious significance for certain native American tribes. Many genes are also rearranged through other transposon-induced modifications such as inversions, deletions, additions, and ectopic recombinations (Bennetzen, Plant Mol Biol 42: 251-69, 2000). Furthermore, plant DNA rearrangements frequently occur through the process of intragenic recombination. For instance, by recombining genes involved in resistance against specific pathogens, plants are able to develop resistance genes with new specificities and, thus, co-evolve with their pathogens (Ellis et al., Trends Plant Sci 5: 373-9, 2000). Another example of intragenic recombination relates to how plants reproduce: plants transition from cross-fertilizing to self-fertilizing by recombining genes involved in self-incompatibility (Kusaba et al., Plant Cell 13: 627-43, 2001). Other processes that promote genome evolution include, for instance, chromosome breakage and interchromosomal recombination.
Enhancing the Nutritional Value of Plants and Food Crops
To modify negative traits such as acrylamide accumulation during processing, glycoalkaloid accumulation, accumulation of undesirable advanced glycation products, CIPC accumulation, low levels of resistant starch, bruise susceptibility, cold-induced sweetening, disease susceptibility, low yield and low quality in crop plants through precise breeding, at least one specific expression cassette is incorporated into a host genome. Three different methods are used to eliminate negative traits: (1) overexpression of genes that prevent the occurrence of negative traits, (2) overexpression of mutated versions of genes associated with negative traits in order to titrate out the wild-type gene products with non-functional proteins, and (3) silencing specific genes that are associated with a negative trait by expressing at least one copy of a leader or trailer fragment associated with that gene in the sense and/or antisense orientation.
One example of an endogenous gene that is associated with a negative trait in potato and can be modified in vitro so that it encodes a non-functional protein is the polyphenol oxidase (PPO) gene. Upon impact injury, the PPO gene product is released from the plastid into the cytoplasm (Koussevitzky et al., J. Biol. Chem., 273: 27064-9, 1998), where it will mediate the oxidation of phenols to create a variety of phenoxyl radicals and quinoid derivatives, which are toxic and/or ultimately form undesirable polymers that leave dark discolorations, or “black spots” in the crop.
Overexpressing a mutant PPO gene that contains a nonfunctional copper-binding domain can lower the activity of all PPO genes that are mainly expressed in tubers and associated organs such as sprouts. The mutations render the polyphenol oxidase protein inactive because it is unable to bind copper. The skilled artisan would know where to make point mutations that would, in this case, compromise the function of a gene product. The applicants identified the copper binding domain in potato PPO by aligning the potato PPO protein sequence with a sweet potato PPO protein sequence (Klabunde et al., Nat. Struct. Biol., 5:1084-90, 1998). Areas of conservation, particularly those containing conserved histidine residues in copper-binding sites, were targets for inactivating the transgene product. Because the almost complete absence of PPO activity in such organs may negatively impact the plant's ability to resist pathogens, the present invention also describes an improved method of only lowering a specific PPO gene that is predominantly expressed in all parts of the mature tuber except for the epidermis. Silencing of this specific PPO gene by using a trailer sequence associated with that gene does not reduce PPO expression in the tuber epidermis, the part of the tuber that is most directly exposed to pathogens attempting to infect.
Enzymatic browning induced by the PPO gene not only reduces the quality of potato tubers; it also negatively affects crop foods such as wheat, avocado, banana, lettuce, apple, and pears.
Other genes that are associated with negative traits and can be silenced by using the leader or trailer sequences associated with those genes include the potato R1 gene and L-type phosphorylase genes. Both genes are involved in the degradation of starch to reducing sugars, such as glucose and fructose, which upon heating participate in the Maillard reaction to produce toxic products such as acrylamide. The present invention demonstrates that a reduction of cold-induced sweetening by lowering R1 or phosphorylase activity leads to a reduction of both non-enzymatic browning and acrylamide accumulation during the frying process of potatoes.
The invention also demonstrates the utility of overexpressing certain native genes in genetically modified crops. Levels of Maillard-reaction products such as acrylamide were reduced significantly by lowering the conversion of sucrose to reducing sugars through overexpression of a newly isolated vacuolar invertase inhibitor gene in potato.
The present invention also predicts that potato tubers displaying either an increased level of invertase inhibitor expression or a reduced level of R1 or phosphorylase expression will not require the intensive treatment with chemical sprout inhibitors such as CIPC prior to storage because their lowered levels of reducing sugars will (1) delay sprouting, and (2) allow storage at lower temperatures, thus further delaying sprouting. The highly reduced CIPC-residue levels, or the absence thereof, further enhances the nutritional value of processed foods derived from plants containing certain modified P-DNAs described here.
Thus, French fries or chips derived from tubers that contain the modified P-DNA will contain strongly reduced CIPC residue levels, further boosting their nutritional value.
The effect of simultaneously downregulating the expression of the PPO and either R1 or phosphorylase genes in potato tubers is synergistic because reducing sugars are not only required for non-enzymatic browning through the Maillard reaction but also for browning mediated by the PPO enzyme. Decreased levels of reducing sugars in transgenic potato tubers will, therefore, also limit PPO activity and black spot bruise susceptibility. Thus, PPO, R1, and phosphorylase genes, and/or the leader or trailer sequences that are associated with these genes, represent DNA segments of interest that can be isolated, modified and reintroduced back into the plant to down-regulate the expression of these genes.
Apart from developing bruise resistance and reduced cold-induced sweetening, there are many other traits that can be introduced through Precise Breeding without using foreign DNA. For instance, disease resistance genes can be isolated from wild potato species and inserted into the genomes of disease susceptible varieties.
The Environmental Benefits of Modified Plants and Crops
As described above, reduced levels of either R1 or phosphorylase result in a reduced phosphorylation of starch. This reduction in starch phosphorylation results in a 90% decrease in phosphate content of potato tubers (Vikso-Nielsen, Biomacromolecules, 2: 836-43, 2001). This will result in a reduction in phosphate levels in wastewaters from potato processing plants, which are currently about 25-40 mg/L. Thus, the use of low-phosphate tubers will reduce the release of phosphates into the environment and help to protect important ecosystems. Furthermore, low-phosphate potatoes may require less phosphate fertilization for optimal growth and yield, which would support a more sustainable agriculture by delaying the depletion of available phosphate resources.
Enhancing the Agricultural Performance of Plants and Food Crops
Apart from reduced bruise susceptibility and reduced cold-sweetening, which are two important processing traits, the present invention also provides salt tolerance, an increasingly important input trait. Some of the modified P-DNA constructs described in the present invention contain a salt tolerance gene as native marker for transformation. Importantly, the utility of this gene is not limited to a screening step in the transformation procedure. Overexpression of the salt tolerance gene in potato plants reduces stress symptoms induced by high salinity soil levels, and will make it possible to grow new varieties containing a modified P-DNA on a growing percentage of agricultural lands that contain salinity levels exceeding the maximum 2 millimhos/cm electrical conductivity levels that are optimal for growing conventional varieties.
Using Regulatory Elements Isolated from a Selected Plant Species or from a Species Sexually Compatible with the Selected Plant Species
Once the leader, gene or trailer has been isolated from the plant species of interest, and optionally modified, it can be operably linked to a plant promoter or similar regulatory element for appropriate expression in plants. Regulatory elements such as these serve to express untranslated sequences associated with a gene of interest in specific tissues or at certain levels or at particular times.
Dependent on the strategy involved in modifying the trait, it may be necessary to limit silencing to a particular region of the plant. The promoter normally driving the expression of the endogenous gene may not be suitable for tissue-specific expression. As described in the section above, stable integration of bacterial or viral regulatory components, such as the cauliflower mosaic virus 35S “super” promoter, can result in unpredictable and undesirable events. Thus, one aspect of the present invention uses promoters that are isolated from the selected host plant species.
In a preferred embodiment of the instant invention, for use in S. tuberosum, the leader or trailer sequences associated with R1, phosphorylase, and PPO genes are operably linked to the granule-bound starch synthase gene promoter (Rohde et al., J Gen & Breed, 44, 311-315, 1990). This promoter has been used frequently by others to drive gene expression and is particularly active in potato tubers (van der Steege et al., Plant Mol Biol, 20: 19-30, 1992; Beaujean et al., Biotechnol. Bioeng, 70: 9-16, 2000; Oxenboll et al., Proc Natl Acad Sci USA, 9: 7639-44, 2000). This promoter may also be used, in a preferred embodiment, for expression of the modified leader or trailer sequences of R1, phosphorylase, and PPO genes.
Alternatively, other potato promoters can be operably linked to sequences of interest from potato. Such promoters include the patatin gene promoter (Bevan et al., Nucleic Acids Res, 14: 4625-38, 1986), or a fragment thereof, that promotes expression in potato tubers, the potato UDP-glucose pyrophosphorylase gene promoter (U.S. Pat. No. 5,932,783) and the promoter of the ubiquitin gene (Garbarino et al., Plant Physiol, 109: 1371-8, 1995).
The transcription of leaders and/or trailers can also be regulated by using inducible promoters and regulatory regions that are operably linked in a construct to a polynucleotide of interest. Examples of inducible promoters include those that are sensitive to temperature, such as heat or cold shock promoters. For instance, the potato ci21A-, and C17-promoters are cold-inducible (Kirch et al., Plant Mol. Biol, 33: 897-909, 1997; Schneider et al., Plant Physiol, 113: 335-45, 1997).
Other inducible promoters may be used that are responsive to certain substrates like antibiotics, other chemical substances, or pH. For instance, abscisic acid and gibberellic acid are known to affect the intracellular pH of plant cells and in so doing, regulate the Rab 16A gene and the alpha-amylase 1/6-4 promoter (Heimovaara-Dijkstra et al., Plant Mol Biol, 4 815-20, 1995). Abscisic acid, wounding and methyl jasmonate are also known to induce the potato pint promoter (Lorberth et al., Plant J, 2: 477-86, 1992).
In another example, some nucleotide sequences are under temporal regulation and are activated to express a downstream sequence only during a certain developmental stage of the plant or during certain hours of the day. For instance, the potato promoter of the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) gene can direct cell-specific, light-regulated expression (Fritz et al., Proc Natl Acad Sci USA, 88: 4458-62, 1991). The skilled artisan is well versed in these exemplary forms of inducible promoters and regulatory sequences.
The use of certain polyadenylation signals may also be useful in regulating expression, by varying the stability of the mRNA transcript. In particular, some polyadenylation signals when operably linked to the '3 end of a polynucleotide cause the mRNA transcript to become accessible to degradation.
Thus, it is possible to regulate expression of a gene by operably linking it with one or more of such promoters, regulatory sequences, 3′ polyadenylation signals, 3′ untranslated regions, signal peptides and the like. According to the instant invention, DNA sequences and regulatory elements such as those described herein, and which will ultimately be integrated into a plant genome, are obtained from DNA of the selected plant species to be modified by the Precise Breeding process of the present invention. That is, DNA sequences and regulatory elements that are derived, isolated and cloned from other species, such as from bacteria, viruses, microorganisms, mammals, birds, reptiles and sexually incompatible plant species are not integrated into the genome of the transformed plant. DNA foreign to the selected plant species genome may be used in the present invention to create a transformation construct, so long as that foreign DNA is not integrated into a plant genome.
Not only does the present invention provide a method for transforming a plant species by integrating DNA obtained from the selected plant species, or from a plant that is sexually-compatible with the selected plant species, it also provides a means by which the expression of that DNA can be regulated. Accordingly, it is possible to optimize the expression of a certain sequence, either by tissue-specific or some other strategy, as previously described.
Using 3′ Terminator Sequences Isolated from a Selected Plant Species
In addition to regulatory elements that initiate transcription, the native expression cassette also requires elements that terminate transcription at the 3′-end from the transcription initiation regulatory region. The transcription termination region and the transcription initiation region may be obtained from the same gene or from different genes. The transcription termination region may be selected, particularly for stability of the mRNA to enhance expression.
This particular element, the so-called “3′-untranslated region” is important in transporting, stabilizing, localizing and terminating the gene transcript. In this respect, it is well known to those in the art, that the 3′-untranslated region can form certain hairpin loop. Accordingly, the present invention envisions the possibility of operably linking a 3′ untranslated region to the 3′ end of a cloned polynucleotide such that the resultant mRNA transcript may be exposed to factors which act upon sequences and structures conferred by the 3′ untranslated region.
A 3′ sequence of the ubiquitin gene can be subcloned from the plant species from which the promoter and transgene were isolated and inserted downstream from a transgene to ensure appropriate termination of transcription. Both exemplary transgenes can be fused to the terminator sequence of the potato Ubiquitin gene (Ubi3) regardless of which promoter is used to drive their expression.
This example demonstrates that T-DNA borders are specific to Agrobacterium. It also shows that plants contain T-DNA border-like sequences, and it provides the sequence of DNA fragments isolated from potato and wheat that are delineated by such border-like sequences.
Conventional transformation systems use Agrobacterium-derived T-DNAs as vehicles for the transfer of foreign DNA from Agrobacterium to plant cells (Schilperoort et al., U.S. Pat. No. 4,940,838, 1990). Although T-DNAs usually comprise several hundreds of basepairs, delineated by a left-border (LB) and right-border (RB) repeat, they can also merely consist of such borders. The T-DNA borders play an essential role in the DNA transfer process because they function as specific recognition sites for virD2-catalyzed nicking reaction. The released single stranded DNA, complexed with Agrobacterial virD2 and virE2, is transferred to plant cell nuclei where it often integrates successfully into the plant genome. All T-DNA borders that have been used for foreign DNA transfer are derived from nopaline and octopine strains of Agrobacterium tumefaciens and A. rhizogenes (Table 2). These borders and often some flanking Agrobacterium DNA are present in thousands of binary vectors including, for example, pPAM (AY027531), pJawohl (AF408413), pYL156 (AF406991), pINDEX (AF294982), pC1300 (AF294978), pBI121 (AF485783), pLH9000 (AF458478), pAC161 (AJ315956), BinHygTOp (Z37515), pHELLSGATE (AJ311874), pBAR-35S (AJ251014), pGreen (AJ007829), pBIN19 (X77672), pCAMBIA (AF354046), pX6-GFP (AF330636), pER8 (AF309825), pBI101 (U12639), pSKI074 (AF218466), pAJ1 (AC138659), pAC161 (AJ315956), pSLJ8313 (Y18556), and pGV4939 (AY147202). Recently, two homologs of T-DNA borders were identified in the chrysopine-type Ti plasmid pTiChry5 (Palanichelvam et al., Mol Plant Microbe Interact 13: 1081-91, 2000). The left border homolog is identical to an inactive border homolog located in the middle of the T-DNA of pTi15955. The right border homolog is unusually divergent from the sequence of functional T-DNA borders. It is therefore unlikely that these homologs are functionally active in supporting DNA transfer from pTiChry5 to plant cells.
Development of a new method that makes it possible to transform plants with only native DNA requires, in the first place, a replacement of the T-DNA including LB and RB. Unfortunately, advanced BLAST searches of public databases including those maintained by The National Center For Biotechnology Information, The Institute for Genomic Research, and SANGER failed to identify any border sequences in plants. It was therefore necessary to consider plant DNA sequences that are similar but not identical to T-DNA borders, designated here as “border-like” (border-like). Examples of plant border-like sequences that were identified in public databases are shown in Table 2. The challenge in trying to replace T-DNA borders with border-like sequences is that border sequences are highly conserved (see Table 2). A large part of these sequences is also highly conserved in the nick regions of other bacterial DNA transfer systems such as that of IncP, PC194, and φX174, indicating that these sequences are essential for conjugative-like DNA transfer (Waters et al., Proc Natl Acad Sci 88: 1456-60, 1991). Because there are no reliable data on border sequence requirements, the entire border seems therefore important in the nicking process. A single study that attempted to address this issue by testing the efficacy of border mutants in supporting DNA transfer is unreliable because negative controls did not appear to function appropriately (van Haaren et al., Plant Mol Biol 13: 523-531, 1989). Furthermore, none of the results of this study were confirmed molecularly. Despite these concerns, two possibly effective border mutants are shown in Table 2 as well.
Based on the homology among border sequences, a T-DNA border motif was identified (Table 2). Although this motif comprises 13,824 variants, many of which may not function—or may be inadequate—in transferring DNA, it represents the broadest possible definition of what a T-DNA border sequence is or may be. This border motif was then used to search publicly available DNA databases for homologs using the “Motif Alignment and Search Tool” (Bailey and Gribskov, Bioinformatics 14: 48-54, 1998) and “advanced BLASTN” (“penalty for nucleotide mismatch”=−1; “expect”=105; Altschul et al., Nucleic Acids Res 25: 3389-3402, 1997). Again, these searches did not identify any identical matches in organisms other than Agrobacterium.
To try and increase the chance of isolating a potato DNA fragment containing border-like sequences that correspond to the border motif, DNA was isolated from 100 genetically diverse accessions (the so-called “core collection,” provided by the US Potato Genebank, Wis.). This Potato DNA was pooled and used as template for polymerase chain reactions using a variety of oligonucleotides designed to anneal to borders or border-like sequences. Amplified fragments were sequence analyzed, and the sequence was then confirmed using inverse PCR with nested primers. One of the potato amplified DNA fragments that was of particular interest contains a novel sequence without any major open reading frames that is delineated by border-like sequences (Table 2) the oligonucleotides. One Each of the border-like termini sequences of this fragment contains at least 5 mismatches with T-DNA borders; the other border-like sequence contains at least 2 mismatches. Although both termini sequences contain one mismatch mismatches with the border motif, they were tested for their ability to support DNA transfer. For that purpose, the fragment was first reduced in size to 0.4-kilo basepairs by carrying out an internal deletion (SEQ ID NO.: 1, see Example 17, and the bold underlined portions of SEQ ID NO. 1 which represent the left (5′-) and right (3′-) termini respectively). The resulting fragment was designated “P-DNA” (plant DNA) to distinguish it from the Agrobacterium-derived T-DNA. A similar fragment was isolated from the genome of the potato variety Russet Ranger, but has not been used for any further experiments.
Based on the divergence between P-DNA and T-DNA borders, the elongase amplification system (Life Technologies) was used with the following degenerate primers to isolate a P- DNA fragment from wheat: 5′-GTTTAGANHNBNATATATCCTGYGA-3′ (Bor-F) (SEQ ID NO. 124), and 5′-TGRCAGGATATATNVNDNTGTAAAC-3′ (Bor-R) (SEQ ID NO. 57). The resulting 825-bp fragment is shown in SEQ ID NO.: 2, and was used to replace the T-DNA of a conventional binary vector. The efficacy of this construct can be tested by inserting an expression cassette for the GUS gene between P-DNA termini, and infecting wheat with an Agrobacterium strain carrying the resulting vector.
This Example demonstrates that, despite structural (sequence divergence) and functional (transformation frequencies) differences between P-DNA termini in SEQ ID NO. 1 and T-DNA borders, a P-DNA they can be used in a similar way as a T-DNA to transfer DNA from Agrobacterium to tobacco cells.
A T-DNA-free vector that can be maintained in both E. coli and A. tumefaciens was obtained by removing the entire T-DNA region of the conventional binary vector pCAMBIA1301 (Cambia, AU). This was accomplished by simultaneously ligating a 5.9 kb SacII-SphI fragment of pSIM1301 with 2 fragments amplified from pCAMBIA1301 using the oligonucleotides pairs: 5′-CCGCGGTGATCACAGGCAGCAAC-3′ (SEQ ID NO. 58) and 5′-AAGCTTCCAGCCAGCCAACAGCTCCCCGAC-3′ (SEQ ID NO. 59), and 5′-AAGCTTGGCTACTAGTGCGAGATCTCTAAGAGAAAAGAGCGTTTA-3′ (SEQ ID NO. 60), and 5′-GCATGCTCGAGATAGGTGACCACATACAAATGGACGAACGG-3′ (SEQ ID NO. 61), respectively.
To make it possible to screen against backbone integration events, an expression cassette comprising the Agrobacterium isopentenyl transferase (IPT) gene driven by the Ubi3 promoter and followed by the Ubi3 terminator (SEQ ID NO.: 3) was inserted as 2.6 kbp SacII fragment into the backbone of the T-DNA-free vector described above, yielding pSIM100-OD-IPT. Transformed plant cells expressing the IPT gene are expected to accumulate cytokinins and grow into abnormal shoots that cannot develop roots.
The 0.4 kb P-DNA fragment described in Example 1 was inserted into pSIM100-OD-IPT to generate pSIM111 (
To test whether pSIM111 can be used to obtain transformed plants carrying P-DNAs (including any sequences the termini in SEQ ID NO. 1 and a sequence located between P-DNA the termini) without the additional vector backbone, a neomycin phosphotransferase (NPTII) gene expression cassette was inserted into the P-DNA of pSIM111 to create pSIM108 (
The efficacy of P-DNA termini in SEQ ID NO. 1 in supporting DNA transfer was tested by comparing transformation frequencies between pSIM108 and a control vector that contained a modified P-DNA with conventional T-DNA borders. This control vector, designated pSIM109, was generated by amplification of the entire P-DNA containing the NPTII gene expression cassette with the oligonucleotide pairs: 5′-ACTAGTGTTTACCCGCCAATATATCCTGTCAGAG-3′ (SEQ ID NO. 62), and 5′-AAGCTTTGGCAGGATATATTGTGGTGTAAACGAAG-3′ (SEQ ID NO. 63). A second control vector that was used for these experiments is the conventional binary vector pBI121 (Genbank accession number AF485783), which contains the same NPTII expression cassette inserted on a regular T-DNA. The binary vectors were introduced into Agrobacterium tumefaciens LBA4404 cells as follows. Competent LB4404 cells (50 μL) were incubated for 5 minutes at 37° C. in the presence of 1 μg of vector DNA, frozen for about 15 seconds in liquid nitrogen (about −196° C.), and incubated again at 37° C. for 5 minutes. After adding 1 mL of liquid broth (LB), the treated cells were grown for 3 hours at 28° C. and plated on LB/agar containing streptomycin (100 mg/L) and kanamycin (100 mg/L). The vector DNAs were then isolated from overnight cultures of individual LBA4404 colonies and examined by restriction analysis to confirm the presence of intact plasmid DNA.
Test transformations of the model plant tobacco were carried out by growing a 10-fold dilution of overnight-grown LBA4404::pSIM108 cells for 5-6 hours, precipitating the cells for 15 minutes at 2,800 RPM, washing them with MS liquid medium (Phytotechnology) supplemented with sucrose (3%, pH 5.7) and resuspending the cells in the same medium to an OD600 nm of 0.2. The suspension was then used to infect leaf explants of 4-week-old in vitro grown Nicotiana tabacum plants. Infected tobacco explants were incubated for 2 days on co-culture medium ( 1/10 MS salts, 3% sucrose, pH 5.7) containing 6 g/L agar at 25° C. in a Percival growth chamber (16 hrs light) and subsequently transferred to M401/agar medium containing timentine (150 mg/L) and kanamycin (100 mg/L). The number of calli per explant that developed within the next 4 weeks is shown in Table 3. Our data demonstrate that P- DNAs delineated by either native termini in SEQ ID NO. 1 or conventional T-DNA borders are about 50% more effective in transforming tobacco than T-DNAs. The increased efficiency of P- DNA transfer may be due to either its different CG content or other unknown structural features of the P- DNA comprising the termini in SEQ ID NO. 1.
This Example demonstrates that a P-DNA DNA comprising the termini in SEQ ID NO. 1 can be used in a similar way as a T-DNA to transfer DNA from Agrobacterium to potato cells.
Potato transformations were carried out by infecting stem explants of 4-week-old in vitro grown Russet Ranger plantlets with Agrobacterium strains according to the following procedure. Ten-fold dilutions of overnight-grown cultures were grown for 5-6 hours, precipitated for 15 minutes at 2,800 RPM, washed with MS liquid medium (Phytotechnology) supplemented with sucrose (3%, pH 5.7), and resuspended in the same medium to an OD600 nm of 0.2. The resuspended cells were then used to infect 0.4-0.6 mm internodal potato segments. Infected stems were incubated for 2 days on co-culture medium ( 1/10 MS salts, 3% sucrose, pH 5.7) containing 6 g/L agar at 22° C. in a Percival growth chamber (16 hrs light) and subsequently transferred to callus induction medium (CIM, MS medium supplemented with 3% sucrose 3, 2.5 mg/L of zeatin riboside, 0.1 mg/L of naphthalene acetic acid, and 6 g/L of agar) containing timentine (150 mg/L) and kanamycin (100 mg/L). After 1 month of culture on CIM, explants were transferred to shoot induction medium (SIM, MS medium supplemented with 3% sucrose, 2.5 mg/L of zeatin riboside,0.3 mg/L of giberelic acid GA3, and 6 g/L of agar) containing timentine and kanamycin (150 and 100 mg/L respectively). After 3-4 weeks, the number of explants developing transgenic calli and/or shooting was counted. As shown in tobaco tobacco, the number of stem explants infected with pSIM 108 that showed calli was higher than those in control experiments with the conventional binary vector pBI121 (Table 3). Shoots that subsequently arose from these calli could be grouped into two different classes. The first class of shoots was phenotypically indistinguishable from control shoots transformed with LBA::pBI121. The second class of shoots displayed an IPT phenotype. Shoots of the latter class were stunted in growth, contained only very small leaves, displayed a light-green to yellow color, and were unable to root upon transfer to hormone-free media. To confirm that shoots with an IPT phenotype contained the IPT gene stably integrated in their genomes, all shoots were transferred to Magenta boxes containing MS medium supplemented with 3% sucrose and timentine 150 mg/L, allowed to grow for 3 to 4 additional weeks, and used to isolate DNA. This plant DNA served as template in PCR reactions with an oligonucleotide pair designed to anneal to the IPT gene: 5′-GTC CAA CTT GCA CAG GAA AGA C-3′ (SEQ ID NO: 122), and 5′-CAT GGA TGA AAT ACT CCT GAG C-3′ (SEQ ID NO: 123). As shown in Table 4, the PCR experiment confirmed a strict correlation between IPT phenotype and presence of the IPT gene. The presence of backbone DNA was also examined in plants obtained from a transformation with pBI121. This was done by performing PCR reactions on DNA isolated from the transformation events with the ‘pBI121 backbone primers’: 5′-CGGTGTAAGTGAACTGCAGTTGCCATG-3′ (SEQ ID NO. 64), and 5′-CATCGGCCTCACTCATGAGCAGATTG-3′ (SEQ ID NO. 65). Amplification of a 0.7 kbp band is indicative for backbone integration. By comparing the data presented in Table 4, it can be concluded that backbone integration frequencies are similar for P-DNA vectors comprising the termini in SEQ ID NO. 1 and T-DNA vectors.
A second PCR experiment was carried out to test whether IPT-free plants did not contain any other backbone sequences. Because the IPT expression cassette is positioned close to the left border-like sequences, the oligonucleotide pair for this experiment was designed to anneal to backbone sequences close to the right border-like sequence: 5′-CACGCTAAGTGCCGGCCGTCCGAG-3′ (SEQ ID NO. 66), and 5′-TCCTAATCGACGGCGCACCGGCTG-3′ (SEQ ID NO. 67). Data from this experiment confirm that plants that are positive for the IPT gene are also positive for this other part of the backbone.
Similar experiments were carried out with the potato variety Russet Burbank. Based on an assessment of IPT phenotypes, the backbone integration frequencies for pSIM108 and pSIM109 were shown to be comparable to those in Russet Ranger (see Tables 4 and 5).
Using conventional transformation methods, this Example demonstrates that overexpressing a novel potato invertase inhibitor gene enhances the processing and health characteristics of potato tubers.
The following primers were designed to amplify a new potato homolog of the tobacco vacuolar invertase inhibitor Nt-inhh1 (Greiner et al., Nature Biotechnology, 17, 708-711, 1999): 5′-AAAGTTGAATTCAAATGAGAAATTTATTC-3′ (SEQ ID NO. 68), and 5′-TTTTAAGCTTTCATAATAACATTCTAAT-3′ (SEQ ID NO. 69). The amplification reaction was performed by mixing the following components: 4 μl plant DNA, 2 μl forward primer (10 pM/ml), 2 μl reverse primer, 25 μl Hot Start Master Mix (Qiagen Catalog Nr. 203443), and 17 μl water. This reaction mix was subjected to the following polymerase chain reaction (PCR) conditions using a PTC-100 thermocycler (MJ Research): (1) 5 minutes at 95° C. (1 cycle), (2) 1 minute at 94° C., 1 minute at 45° C. and 4 minutes at 72° C. (35 cycles), and (3) 10 minutes at 72° C. (1 cycle). The total product was loaded on a 0.8% agarose gel, and a 540 base pair band was purified from gel using QIAquick Gel Extraction Kit (Qiagen, Calif.). This purified fragment was then ligated into pGEM-T Easy (Promega, Wis.) and transformed into E. coli DH5-alpha using Max Efficiency Competent Cells (GibcoBRL, MD). Sequence analysis of recombinant plasmid DNA isolated from transformed DH5-alpha revealed the presence of a single open reading frame consisting of 543 base pairs that encodes for a putative 181-amino acid protein (SEQ ID NO.: 5); clustal-aligment revealed 70% homology to Nt-inhh (
Although the St-inh1 gene is present in unmodified potato tubers, its expression level is inadequate for full inhibition of invertase and reduced cold-induced sweetening. To increase the storage characteristics of potato, the St-inh1 gene was fused to a new tuber-enhanced promoter of the granule-bound starch synthase (GBSS) gene, which is known to promote high levels of gene expression in tubers. The GBSS promoter was isolated from the potato cultivar Russet Ranger by carrying out a PCR reaction using the forward primer 5′-GAACCATGCATCTCAATC-3′ (SEQ ID NO. 70) and the reverse primer 5′-GTCAGGATCCCTACCAAGCTACAGATGAAC-3′ (SEQ ID NO. 71). Sequence analysis of the amplified product cloned in pGEM-T demonstrated that this new promoter contains 658 basepairs (SEQ ID NO.: 6). The resulting promoter/gene fusion was then ligated to the 3′ regulatory sequence of the potato ubiquitin gene (UbiT; SEQ ID NO.: 7), thus ensuring appropriate termination of transcription of the invertase inhibitor gene.
This expression cassette was inserted between T-DNA borders of a binary vector, and the resulting vector pSIM320 was used to transform Russet Ranger as described above. Three cuttings of nine independent transgenic lines were planted in soil and grown for four weeks in a growth chamber (11 hrs light; 20° C.). At least 3 minitubers were then harvested from each line and transferred to a refrigerator set at 4° C. to induce cold-sweetening. After 4 weeks, the glucose levels in these cold-stored minitubers were determined by using either an Accu-Chek meter and test strips (Roche Diagnostics, IN) or a glucose oxidase/peroxidase reagent (Megazyme, Ireland). These levels were compared with the average glucose levels in both 6 untransformed lines and 6 “vector control” lines transformed with a pSIM110-derived vector lacking the invertase inhibitor gene. As shown in Table 6, three transgenic lines accumulated less than 40% of the glucose in “vector control” lines demonstrating that the potato invertase inhibitor homolog is functionally active.
The following experiment showed that the amount of reducing sugars present in tubers correlates with acrylamide production during tuber processing. Russet Ranger potato tubers were freshly harvested from the field and stored at 4° C. to induce cold-sweetening; control tubers were stored at 18° C. After 4 weeks, glucose levels were determined in both groups of tubers. Subsequently, tubers were washed, blanched for either 8 minutes or 12 minutes at 165° F., cut into 0.290×0.290 shoestring strips, dipped in a 1% sodium acid pyrophosphate solution at 160° F., dried at 160° F. until 14±2% dryer weight loss is achieved, fried at 390° F. for 40 seconds to attain 64±2% first fry moisture, and frozen for 20 minutes at −15° F., shaking the tray 2-3 times in the first 6 minutes. The resulting French fries were then analyzed for acrylamide levels by Covance laboratory (WI). As shown in Table 7, the glucose levels in tubers stored at 18° C. were below the detection level of 0.1 mg/g whereas cold-stored tubers contained on average 3.4 mg/g glucose. This table also shows that fries produced from the latter potatoes contain about 10-fold higher levels of acrylamide than fries produced from potatoes stored at 18° C. Even by using a shorter blanch time for 18° C.-stored potatoes than for 4° C.-stored potatoes to produce fries with a similar color (color ids of 78 and 71, respectively), a 5-fold difference in acrylamide accumulation was obtained (Table 7). Thus, there appears to be a straight correlation between the amount of reducing sugars such as glucose in tubers and the accumulation of acrylamide in fries derived from these tubers.
To determine whether the reduced glucose levels in pSIM320 lines would limit the processing-induced accumulation of acrylamide, cold-stored pSIM320 minitubers were processed by cutting into wedges, blanching for 8 minutes, dipping in 0.5% SAPP for 30 seconds, drying for 4.5 minutes at 160° F., frying for 40 seconds at 380° F., freezing for 15 minutes at −15° F., and finally drying for 3 minutes and 10 seconds at 160° F. The processed material was then shipped to Covance laboratory for acrylamide determinations. As shown in Table 6, French fries obtained from minitubers with the lowest amounts of glucose accumulated the lowest levels of acrylamide. A 40% reduction in glucose levels in lines “320-2” and “320-4” is associated with a 5-fold reduction in acrylamide levels.
Using conventional transformation methods, this Example demonstrates that a novel leader sequence associated with the potato R1 gene can be used effectively to enhance the processing and health characteristics of potato tubers. It also predicts that a novel trailer associated with that same gene can be exploited in the same way.
As an alternative to the leader-based approach, expression cassettes that contained both a sense and antisense copy of the trailer sequence associated with R1 were generated. This trailer was obtained by performing a reverse transcription polymerase chain reaction (RT-PCR) on total RNA isolated from microtubers of the potato cultivar Russet Ranger. Complementary DNA was generated using the Omniscript RT Kit (Qiagen, Calif.) and then used as a template for a PCR reaction with Hot start DNA polymerase (Qiagen, Calif.) with the gene-specific reverse primer R1-1 (5′-GTTCAGACAAGACCACAGATGTGA-3′ SEQ ID NO: 114). Sequence analysis of the amplified DNA fragment, cloned in pGEM-T demonstrated that the trailer associated with R1 consists of 333 basepairs (SEQ ID NO.: 16). The sense and antisense copies of the trailer were separated by either the Ubi intron or the GBSS spacer- and sandwiched between GBSS promoter and Ubi3 terminator (
To test the efficacy of the R1-associated leader in limiting acrylamide production, the expression cassette shown in
As an alternative to the leader-based approach, expression cassettes that contained both a sense and antisense copy of the trailer sequence associated with R1 were generated. This trailer was obtained by performing a reverse transcription polymerase chain reaction (RT-PCR) on total RNA isolated from microtubers of the potato cultivar Russet Ranger. Complementary DNA was generated using the Omniscript RT Kit (Qiagen, Calif.) and then used as a template for a PCR reaction with Hot start DNA polymerase (Qiagen, Calif.) with the gene-specific reverse primer R1-1 (5′-GTTCAGACAAGACCACAGATGTGA-3′). Sequence analysis of the amplified DNA fragment, cloned in pGEM-T demonstrated that the trailer associated with R1 consists of 333 basepairs (SEQ ID NO.: 16). The sense and antisense copies of the trailer were separated by either the Ubi intron or the GBSS spacer- and sandwiched between GBSS promoter and Ubi3 terminator (
Glucose and acrylamide levels can be determined as described above. Tubers displaying about 50% or greater reductions in glucose concentrations are expected to also accumulate about 50% less acrylamide during the frying process. The improved health and storage characteristics of modified plants can be confirmed in mature field-grown tubers.
Phosphate levels in potato tubers can be determined by using AOAC Method 995.11 Phosphorus (Total) in Foods (45.1.33 Official Methods of Analysis of AOAC International, 17th Edition). Samples are prepared by dry ashing in a muffle furnace followed with an acid digestion. The dissolved samples are then neutralized and treated with a molybdate-ascorbic acid solution and compared to a series of phosphorus standards (treated similarly). A dual beam spectrophotometer would be used for the colorimetric analysis at 823 nanometers. A significant decrease in phosphate content, which is beneficial for the environment, is expected.
Using conventional transformation methods, this Example demonstrates that a novel leader sequence associated with the potato L-alpha glucan phosphorylase gene can be used to effectively enhance the processing and health characteristics of potato tubers.
Previously, it was shown that cold-induced sweetening can be reduced through antisense expression of 0.9-kb fragments derived from alpha glucan phosphorylase genes (Kawchuk et al., U.S. Pat. No. 5,998,701, 1999). However, the antisense expression of these relatively large DNA fragments is undesirable because they contain new and uncharacterized open reading frames that may impact the nutritional quality of foods if expressed in transgenic plants (Table 1).
As a safer approach to the one described above, small leader and trailer sequences that are associated with a L-type glucan phosphorylase gene were isolated from RNA of mature tubers. The primer pair used for this purpose is: 5′-GGATCCGAGTGTGGGTAAGTAATTAAG-3′ (SEQ ID NO. 72), and 5′-GAATTCTGTGCTCTCTATGCAAATCTAGC-3′ (SEQ ID NO. 73). The resultant leader sequence of 273 bp was amplified and is shown in SEQ ID NO.: 21. Similarly, the “direct” primer, 5′-GGAACATTGAAGCTGTGG-3′ (SEQ ID NO. 74), was used with an oligo-dT primer to amplify a 158 bp “trailer sequence” that is associated with the L-type phosphorylase gene (SEQ ID NO.: 22).
Expression cassettes were then designed using these trailer or leader sequences to modify the expression of L-type phosphorylase gene and, in so doing, lowering acrylamide levels in fried products by limiting starch mobilization. These cassettes were constructed in a similar way as described in Example 5, and are depicted in
Four lines that displayed at least 50% reduced glucose concentrations (lines 216-2, 216-5, 216-10, and 216-21) were used to assess processing-induced acrylamide levels. Although acrylamide levels in fried tubers derived from the first three lines were similar to those of controls, French fries that were derived from line 216-21 accumulated only 45% of the wild-type acrylamide levels (136 vs. 305 parts per billion). These results confirm the experiments described in Example 4 for tubers overexpressing the potato invertase inhibitor gene, in that relatively large reductions in glucose (and fructose) concentrations are needed to limit the heating-induced acrylamide accumulation in cold-stored minitubers. Because silencing of the phosphorylase gene is expected to be more effective in mature “216” tubers, reductions in acrylamide levels are also anticipated to be more pronounced in the French fries produced from such tubers. The improved health and storage characteristics of modified plants can be confirmed in mature tubers.
Using conventional transformation methods, this Example demonstrates that a modified polyphenol oxidase gene lacking a functional copper-binding site can be used effectively to reduce bruise susceptibility in tubers.
Previously, it was shown that black spot bruise susceptibility can be reduced through antisense expression of the 1.8-kb PPO gene (Steffens, U.S. Pat. No. 6,160,204, 2000). However, expression of the reverse complement of this large gene is undesirable because it contains new and uncharacterized open reading frames encoding peptides consisting of more than 100 amino acids, which may potentially impact the nutritional quality of foods (Table 1). As a safer approach to the one described above, the PPO gene was modified to encode a non-functional protein.
The wild-type potato PPO gene was isolated from Russet Ranger by using a polymerase chain reaction (PCR) method. First, genomic DNA was isolated from sprouts of Russet Ranger. The potato PPO gene was then amplified from the potato genomic DNA using DNA polymerase and oligonucleotide primers: 5′: CGAATTCATGGCAAGCTTGTGCAATAG-3′ (PPO-F) (SEQ ID NO. 75), and 5′-CGAATTCTTAACAATCTGCAAGACTGATCG-3′ (PPO-R) (SEQ ID NO. 76). These were designed to complement the 5′- and 3′-ends of the potato PPO gene. The amplified 1.6 kb fragment was cloned into a pGEM-T EASY vector (Promega) and confirmed to represent a functional PPO gene by sequence analysis (SEQ ID NO.: 27).
The copper binding domain in potato PPO was identified by aligning this protein with a sweet potato PPO protein that was shown to contain conserved Cysteine (Cys) residue at position 92, Glutamine residue (Glu) at position 236, and Histidine (His) residues at positions 88, 109, 118, 240, 244 and 274 coordinating the two active site coppers (Klabunde et al., Nature Structural Biol., 5: 1084-1090, 1998). These Cys, Glu, and His residues are also present in potato PPO.
The inactive PPO gene was created by using a PCR mutation replacement approach. Three fragments were amplified by Proof Start Taq DNA Polymerase (Qiagen) using 3 pairs of primers and wild-type Russet Ranger PPO as a template. The sequences of the first pair, designated P1-F and P2-R, respectively, are: 5′-GAGAGATCTTGATAAGACACAACC-3′ (SEQ ID NO. 77), and 5′-CATTACC1ATAAGCC2CAC3TGTATATTAGCTTGTT GC-3′ (SEQ ID NO. 78) (1: “A” to “C” mutation, resulting in Cysteine to Glycine substitution at position 186; 2: “A” to “C” mutation, resulting in Cysteine to Tryptophan substitution at position 183; 3: “A” to “C” mutation, resulting in Histine to Glutamine substitution at position 182). The sequences of the second pair, designated P3-F and P4-R, respectively, are 5′-GTGCTTATAGAATTGGTGGC-3′ (SEQ ID NO. 79), and 5′-TAGTTCCCGGGAGTTCAGTG-3′ (SEQ ID NO. 80). The sequences of the third pair, designated P5-F and P6-R, respectively, are 5′-CTCCCGGGAACTATAGG4AAACATTCCTCT5CGGT CCTGTCCACATCTGGTC-3′ (SEQ ID NO. 81) and 5′-GTGTGATATCTGTTCTTTTCC-3′ (SEQ ID NO. 82) (4: “A” to “G” mutation, resulting in Glutamine to Glycine substitution at position 326; 5: “A” to “T” mutation, resulting in Histine to Leucine substitution at position 330).
An 80 bp fragment was amplified using primer P1-F and P2-R and digested with BglII. This fragment contains one sticky end (BglII) and one blunt end, and carries three mutations in copper binding site I. A 0.4 kb fragment amplified using primer P3-F and P4-R and digested with XmaI contains one blunt end and one sticky end (XmaI). A 0.2 Kb fragment was amplified using primer P5-F and P6-R and digested with XmaI and EcoRV. This third fragment with a sticky end (XmaI) and a blunt end (EcoRV) has two mutations in copper binding site II. The BglII and EcoRV fragment from cloned wild-type potato PPO was then replaced with the above three ligated PCR amplified fragments. The presence of a total of 5 point mutations in the modified PPO gene was confirmed by sequence analysis (SEQ ID NO.: 28). To create an expression cassette for modified PPO (mPPO), the following four fragments were simultaneously ligated together: (1) a BamHI-HindIII fragment containing the GBSS promoter, (2) a HindIII-SacI fragment containing mutant PPO, (3) a SacI-KpnI fragment containing the Ubi-3 terminator, and (4) plasmid pBluescript, digested with KpnI and BamHI. This expression cassette was then inserted between borders of a binary vector to create pSIM314.
The efficacy of the mPPO gene expression cassette was assessed by transforming Russet Ranger stem explants with pSIM314. Nodal cuttings of transgenic plants containing this expression cassette were placed on MS medium supplemented with 7% sucrose. After a 5-week incubation period in the dark at 18° C., microtubers were isolated and assayed for PPO activity. For this purpose, 1 g of potato tubers was pulverized in liquid nitrogen. This powder was then added to 5 ml of 50 mM MOPS (3-(N-morpholino) propane-sulfonic acid) buffer (pH 6.5) containing 50 mM catechol, and incubated at room temperature with rotation for about 1 hour. The solid fraction was then precipitated, and the supernatant transferred to another tube to determine PPO activity by measuring the change of OD-410 over time. As shown in Table 10, microtubers isolated from some of the transgenic lines displayed a significantly reduced polyphenol oxidase activity compared to either untransformed controls or controls transformed with a construct not containing the mutant PPO gene. The strongest reduction in PPO activity was observed in lines “314-9”, “314-17”, and “314-29”. To test whether expression of the mutant PPO gene also reduced PPO activity in minitubers, rooted plantlets of transgenic lines were planted in soil and incubated in a growth chamber for 4 weeks. A PPO assay on isolated minitubers demonstrated that reduced PPO activity in microtubers correlated in most cases with reduced activities in minitubers (Table 10). Transgenic lines displaying a reduced PPO activity can be propagated and tested both in the greenhouse and the field to confirm the “low bruise” phenotype in mature tubers. Because micro- and minitubers express a variety of polyphenol oxidases, some of which share only limited sequence homology with the targeted polyphenol oxidase that is predominantly expressed in mature tubers, an even more profound reduction of PPO activity may be anticipated in the mature tubers of lines such as “314-9” and “314-17”. The data indicate that overexpression of a functionally inactive PPO gene can result in reduced bruise susceptibility. The improved health and storage characteristics of modified plants can also be confirmed in mature field-grown tubers.
Using conventional transformation methods, this Example demonstrates that a novel trailer sequence associated with the potato PPO gene can be used effectively to reduce bruise susceptibility in tubers.
Reverse transcription PCR was used to also isolate the trailer sequence associated with the PPO gene expressed in potato tubers. The primers for the first PCR reaction were PPO-1 (5′-GAATGAGCTTGACAAGGCGGAG-3′, (SEQ ID NO. 83)) and oligo-dT; primers for a second nested PCR reaction were PPO-2 (5′-CTGGCGATAACGGAACTGTTG-3′, (SEQ ID NO. 84)) and oligo-dT. Sequence analysis of the amplified DNA fragments cloned into pGEM-T revealed the presence of a 154-bp trailer (SEQ ID NO.: 29). A sense and antisense copy of this trailer, separated by the Ubi intron, was then fused to the GBSS promoter and Ubi3 terminator as described above to generate an expression cassette shown in
Increasing the amylose/amylopectin ratios in tubers can further enhance the nutritional value of potato products. One method that makes it possible to increase amylose content is based on the antisense expression of genes encoding for the starch branching enzyme (SBE) I and II (Schwall et al., Nature Biotechnology 18: 551-554, 2000). The disadvantages of this method are that (1) the efficiency of simultaneously silencing two different genes through exploitation of antisense technologies is very low, (2) the antisense expression of the relatively large SBE-I and SBE-II gene sequences results in the undesirable expression of open reading frames (Table 1) (3) corresponding constructs that harbor the two antisense expression cassettes are unnecessarily large and complex, thus, increasing chances of recombination and lowering transformation frequencies.
Our approach to increase amylose content in potato is based on the expression of the trailer sequences that are associated with both genes. These trailers (SEQ ID No.:34 and 35) were isolated with the primer pairs 5′-GTCCATGATGTCTTCAGGGTGGTA-3′ (SEQ ID NO. 85), and 5′-CTAATATTTGATATATGTGATTGT-3′ (SEQ ID NO. 86), and 5′-ACGAACTTGTGATCGCGTTGAAAG-3′ (SEQ ID NO. 87), and 5′-ACTAAGCAAAACCTGCTGAAGCCC-3′ (SEQ ID NO. 88). A single promoter drives expression of a sense and antisense fusion of both trailers, separated by the Ubiquitin-7 intron, and followed by the Ubiquitin-3 terminator. The size of the entire expression cassette is only 2.5-kb.
This Example demonstrates that plants can be transformed effectively without to need for stable integration of selectable marker genes.
This method is the first to take advantage of the phenomenon that DNAs targeted to the nuclei of plant cells often fails to subsequently integrate into the plant cell's genome. The inventors made the surprising discovery that it is possible to select for cells that temporarily express a non-integrating T-DNA containing a selectable marker gene by placing infected explants for 5 days on a plant medium with the appropriate selective agent. A second phenomenon that was applied to develop the current method is that T-DNAs from different binary vectors often target the same plant cell nucleus. By using two different binary vectors, one containing the selectable marker on a T-DNA, and the other one carrying a T-DNA or P-DNA with the actual sequences of interest, it was possible to apply a transient selection system and obtain populations of calli, shoots or plants, a significant portion of which represents marker-free transformation events.
A conventional binary vector designated pSIM011 was used to represent the vector with the “sequence of interest”, which is, in this test case, an expression cassette for the beta glucuronidase (GUS) gene located on a conventional T-DNA. The second binary vector that was used for these experiments contains an expression cassette comprising the neomycin phosphotransferase (NPTII) gene driven by the strong promoter of the Ubiquitin-7 gene and followed by the terminator sequences of the nopaline synthase (nos) gene between the borders of the T-DNA of a pSIM011-derivative.
Surprisingly, a strong level of transient NPTII gene expression levels could also be obtained by replacing the nos terminator with the terminator of the yeast alcohol dehydrogenase 1 (ADH1) gene (Genbank accession number V01292, SEQ ID NO. 56). This finding is interesting because the yeast ADH1 terminator does not share homology with any plant terminator. Importantly, it should be noted here that many yeast terminators do not function adequately in plants. For instance, almost no GUS gene expression was observed in a similar experiment as described above with the GUS gene followed by the yeast iso-1-cytochrome c (CYC1) terminator (Genbank accession number SCCYT1). An improved vector carrying the selectable marker gene NPTII was generated by replacing the nos terminator with the yeast ADH1 terminator. The binary vector containing a selectable marker gene for transient transformation is designated “LifeSupport” (
Potato stem explants were simultaneously infected with two A. tumefaciens LBA4404 strains containing pSIM011 and LifeSupport, respectively. A 1/10 dilution of overnight-grown cultures of each strain were grown for 5-6 hours before they were precipitated, washed and resuspended an OD600 nm of 0.4 as described in Example 3. The resuspended cells were then used to infect 0.4-0.6 cm internodal potato segments at a final density of each bacteria of 0.2 (OD600 nm). Infected stems were treated as in Example 3 with a main difference: the selection with kanamycin was limited to the first 5 days of culture on callus induction medium. Then, explants were allowed to further develop in fresh CIM and SIM containing only timentine 150 mg/L but no selective antibiotic. Within about 3 months from the infection day leaves from shoots derived from calli developed in 40-60% of the infected stems were both tested for GUS expression and PCR analyzed to identify events that contained the sequences of interest but no marker gene. As shown in Table 12, 11% of shoots represented marker-free transformation events.
The two-strain approach described above was also used to transform tobacco. Shoots that developed within about 2 months were GUS assayed and PCR analyzed. The high frequency of marker-free transformation events identified (18%) implies that the developed method is applicable to plant species other than potato (Table 12).
Importantly, sequential rather than simultaneous infection with the two different Agrobacterium strains resulted in an increase in the efficiency of marker-free transformation. The surprising effect of sequential infections was discovered by infecting potato stem explants with the Agrobacterium strain containing pSIM011, placing the infected explants on co-cultivation plates for 4 hours, and then re-infecting them with the LifeSupport vector. The doubly infected explants were treated as previously described in this example. As shown in Table 13, the lag time of 4 hours between the two different infections resulted in a 2-fold increased frequency of marker-free transformation events in potato.
This Example demonstrates the efficacy of precise breeding. The health and agronomic characteristics of potato plants are enhanced by inserting potato genetic elements (see Examples 1, 4, and 7) into potato, using marker-free transformation (Example 10).
A binary vector containing two expression cassettes for the invertase inhibitor and mutant polyphenol oxidase genes inserted between P-DNA the termini in SEQ ID NO. 1, designated pSIM340 (
This Example demonstrates that the efficiency of precise breeding methods can be increased by selecting against stable integration of LifeSupport T-DNAs using the bacterial cytosine deaminase gene.
The previous example demonstrates that the efficiency of marker-free transformation is several-fold lower with a modified P-DNA the DNA in pSIM340 than with a conventional T-DNA. To improve the efficiency of generating shoots only containing a modified P-DNA the DNA in pSIM340, an expression cassette for a suicide gene fusion comprising the bacterial cytosine deaminase (codA) and uracil phosphoribosyltransferase (upp) genes (InvivoGen, CA) was inserted between T-DNA borders of the LifeSupport vector, generating pSIM346 (
An even greater increase in the efficiency of marker-free transformation was obtained by using the LifeSupport vector pSIM350 (
Efficiencies can be further increased by not infecting explants simultaneously with pSIM340 and pSIM350 but sequentially. By infecting the explants with pSIM340 and re-infecting them with pSIM350 after 4 hours, marker-free transformation frequencies are expected to be approximately 30-40%.
This Example demonstrates that the efficiency of precise breeding methods marker-free transformation can be increased by impairing integration of the LifeSupport T-DNA into the plant genome using an omega-mutated virD2 gene.
It has been shown that the omega domain of the Agrobacterium protein vird2 is important for the ability of that protein to support T-DNA integration into plant genomes (Mysore et al., Mol Plant Microbe Interact 11: 668-83, 1998). Based on this observation, modified LifeSupport vectors were created that contain an expression cassette for an omega-mutated virD2 protein inserted into the SacII site in their backbone sequences. The expression cassette was obtained by amplifying a 2.2-kb DNA fragment from plasmid pCS45 (courtesy of Dr. Walt Ream—Oregon State University, OR, USA—, SEQ ID NO.: 36). A LifeSupport-derivative carrying this expression cassette, designated pSIM401Ω (
Efficiencies are further improved by increasing the size of the LifeSupport T-DNA from 3.7 kb (in pSIM401Ω) to 8.1 kb (in the pSIM401Ω-derivative designated pSIM341Ω;
A further improvement can be obtained by infecting explants sequentially rather than simultaneously with pSIM340 and LifeSupport. In a similar way as described in Example 10, the frequency of plants that only contain a modified P-DNA the DNA of pSIM340 can be about doubled by infecting the explants with pSIM340 and re-infecting them with LifeSupport after 4 hours.
This Example demonstrates that high frequencies of marker-free transformation can also be obtained by using a single Agrobacterium strain that contains both the P-DNA vector comprising the termini in SEQ ID NO: 1 and LifeSupport.
Two compatible binary vectors were created that can be maintained simultaneously in Agrobacterium. Instead of using this system to stably integrate two T-DNAs sequences carrying the DNA-of-interest and a marker gene, respectively (Komari et al. U.S. Pat. No. 5,731,179, 1998), it is intended for integration of only the modified P-DNA DNA-of-interest.
The first vector, designated pSIM356, contains an expression cassette comprising the GUS gene driven by the Ubi7 promoter and followed by UbiT inserted between P-DNA termini in SEQ ID NO: 1. The backbone portion of this vector contains bacterial origins of replication from pVS1 and pBR322, a spectinomycin resistance gene for bacterial selection, and an expression cassette for the IPT gene to enable selection against backbone integration in plants (
The concept of increasing marker-free transformation frequencies using pSIM356 and pSIM363 was tested in 100 tobacco shoots. As shown in Table 16, about 19% of regenerated shoots contained the DNA of interest without marker gene. An increase in marker-free transformation efficiency was also found by applying this 1-strain approach to potato. Nine of 60 independent shoots tested (15%) contained the pSIM340 T-DNA and lacked the LifeSupport T-DNA (Table 16).
The 1-strain approach can be combined with the method described in Example 12 to couple a positive selection for transient marker gene expression with a negative selection against stable integration of the codA gene. For this purpose, the LifeSupport vector pSIM365 was developed (
Apart from transforming crop plants with P-DNAs that only contain the desirable sequences to introduce beneficial traits, the present invention also provides a method of transforming such plants with P-DNAs that contain an additional native marker gene. Our novel and native marker genes of choice are potato homologs of the Arabidopsis vacuolar Na+/H+ antiporter gene and alfalfa alfin-1 gene. Expression of these genes do not only allow the identification of transformation events, but also provides salt tolerance to transformed plants. High salinity levels in an increasing acreage of agricultural land will therefore less affect potato plants containing the salt tolerance marker.
Two versions of a vacuolar Na+/H+ antiporter homolog, designated Pst (Potato salt tolerance) were amplified from cDNA of a late blight resistant variety obtained from the US Potato Genbank (WI), designated “LBR4”, using the oligonucleotide pair 5′-CCCGGGATGGCTTCTGTGCTGGCT-3′ (SEQ ID NO. 89) and 5′-GGTACCTCATGGACCCTGTTCCGT-3′ (SEQ ID NO. 90). Their sequences are shown in SEQ ID NO.:37 and 38. A third gene (SEQ ID NO.:39) with homology to allin-1 was amplified from LBR4 potato DNA using the primers 5′-CCCGGGTATGGAAAATTCGGTACCCAGGACTG-3′ (SEQ ID NO. 91) and 5′-ACTAGTTAAACTCTAGCTCTCTTGC-3′ (SEQ ID NO. 92). The efficacy of the Pst genes to function as transformation marker was assessed by inserting a fusion with the Ubi7 promoter between conventional T-DNA borders of a modified pSIM341 vector. After a transient selection period, kanamycin-resistant cells are allowed to proliferate and develop shoots. These shoots are then transferred to media that contain 100-150 mM sodium chloride. Salt-tolerant shoots represent transformation events that contain the T-DNA of the modified pSIM341.
A newly isolated tuber-specific promoter can replace the GBSS promoter used to develop the expression cassettes described in previous examples. This promoter was isolated from the genome of Russet Burbank potato plants by using the inverse polymerase chain reaction with primers specific for a potato proteinase inhibitor gene (Genbank Accession D17332) (SEQ ID NO. 39). The efficacy of the PIP promoter was tested by creating a binary vector that contains the GUS gene driven by this promoter and an expression cassette for the NPTII marker gene. A similar construct with the PIP promoter replaced by the GBSS promoter was used as control. Transformed shoots were obtained by infecting stem explants with Agrobacterium strains carrying the binary vectors, co-cultivation for 2 days, and selection on CIMTK medium for 2 months. These shoots were transferred to new media to induce root formation, and then planted into soil. Tubers can be assayed for GUS expression after a 3-month growth period in the green house.
Apart from pSIM340, many other vectors can be used to improve potato plants by transforming them with modified P-DNAs. Two of such vectors contain an expression cassette for a sense and antisense copy of the trailer associated with a PPO gene that is expressed in all tuber tissues except for the epidermis (see Example 8). Vector pSIM370 contains an additional expression cassette for a sense and antisense copy of the leader associated with phosphorylase gene (see Example 6). Vector pSIM371 contains a third expression cassette for the potato alfin-1 homolog (
A third alternative vector, designated pSIM372, contains both an expression cassette for the potato alfin-1 homolog, and an expression cassette for a sense and antisense copy of a fusion of the PPO-associated trailer, R1-associated leader, and phosphorylase-associated leader.
The preferred LifeSupport vector for a 1-strain approach is pSIM365. For a 2-strain approach, the preferred vector is pSIM367, which contains expression cassettes for both NPTII and codA between T-DNA borders, and an additional expression cassette for omega virD2 in the plasmid backbone (
Potato stem explants are infected with 1 strain carrying both pSIM365 and any of the vectors pSIM370, 371, and 372, or sequentially with 2 strains carrying pSIM366 and any of the preferred vectors-of-interest, respectively. After a 2-day co-cultivation and a 5-day transient selection period, the explants are transferred to media for proliferation/regeneration and elimination of Agrobacterium. Thirty days later, explants are transferred again to the same media but now also containing 5-FU to eliminate events containing LifeSupport T-DNAs. Shoots that subsequently arise on calli are transferred to regeneration media that may contain 100-200 mM salt to screen for salt tolerant events. The IPT-negative shoots are allowed to root and develop into mature plants. A large proportion of these plants (10%-100%) are predicted to represent marker-free and backbone-free plants containing a P-DNA with nucleotide sequences of interest stably integrated into their genomes.
TABLE 1
Potentially expressed uncharacterized peptides in antisense potato lines
Gene (size of
fragment used)
Predicted peptides encoded by ORFS in reverse-complemented DNA
R1 (1.9-kb)
MSSTSNVGQD CLAEVTISYQ WVGRVINYNF FLLIHWYTVV EASTGITFQI FPIGIRSEDD RSFYEKADRF
AWVT
MSSESTFSKT PNGRATDVGI PTEEGTFPFR YAILRDLAPT ISLVNSSADI A
MSEGVGFKSK ILPSFAWRSA NILGSKHVAK QTFPFLARTE TCERTSGMSG VIRATAPSGI SSSPLTDFAT
KIVGFS
GLTP (1-kb)
VCSPALKADK SKSADGTCVD HSRRLIVVLV LYPGMGTSYA TAFISSPPIQ YLFPSDPVET FP
MLGSLVLPKS PENRKQAVPN PHFQEQHLVP EKPHFLDCGQ GFSKLPQMHQ
MVNFLTQGIV DMETAFGSPK MGGFGKEQFG ACVSRSEMDE SGIGAVMVEQ VCSICSRHFV LSMQI
GHTP (0.9-kb)
MLEGSMWPWN QESMKRAFLN HHFLMLHLFP AQRPPQAADP VCLKHQHMHC GCLSFQLHLS KLAPGDTPLI
SSMFALD
MKLCSSIILS IIKQKQVEIL RACFGFPETK TISVFSSVSW NWHIICKSL
MTKKPDRKDN IMPYNFPGTK FLQPIFRNFF LPSLCDKLLK KSISVPQAIT PCWKVQCGHG IKKA
PPO (1.8-kb)
TILKLDLHTF NGHFFTASFW NQSHRNSIFI FQSNILQQFS YRQLESNTGN MISITSMNM RQASITPCKL
RLIKLICIHS LVHVQKHIEP YIVPIIIRYF IECQYLLLLI FLLCCP
MKGKEKPREM NLQFFTTNFV STVAISTMNI SLLFKAKRVK GVFIKFPHST RSQLILGYVL LIRRMSRGAD
AEFSHRRELV VRNTIDLIGY RRATTVYYIN TFFYMGSTTR LEIRRWYRCS SR
MEWALARNRI PFFYCPNSLR TSHGKGYDFH RRKRIQSSTN LYLLNPFFSR QLISIHSTSC PHWHGGSKKS
DLNRVSRNYP CLHRFFDEVC HRSRCEPEYE GCFQ
SBE A (1.2-kb)
MNNITHSPIL IPFLEQLNPF ISNCHMQPIV KANTPILNGN TKCRHSANIF TNGNCIWEKP MNKIVDQHQI
HNSIHISCES KVFLVVPSES HR
MKFRYPSPPN PIVTSLIILC NAIPRSINDV DGLSRAIKSY ISLSISQNAI VLSPTRA
SBE B (2.6-kb)
MVNIMTSSSM ATKFPSITVQ CNSVLPWQVT SNFIPFVCVL WVEVEYKYQV TTFKHNNLII IIHAAYYLFS
MAKLVTHEIE VPLSSQGHCE KMDHLVKRNS SINNRRSICQ ARHARIHLFV H
MFETKLNSGV VWNDWLTVNI RNSNTPNTKL VLLHHVVRTV PSIEIANNFV FLSSRSPFTI DYATIFPVES
KF
MLYTSLYISY LSNSMLLPSW TNLHHSYSLN NLSTYLGLPL PGGNQNQFLP QKQAGQGPAY QKHLRQ
TABLE 3
Transformation efficiency
Calli/tobacco leaf
Calli/potato stem
Binary vector
explant ± SE
explant ± SE
pBI121
7.8 ± 0.6
0.31 ± 0.10
pSIM108
10.2 ± 0.6
0.59 ± 0.07
pSIM109
12.8 ± 0.6
0.47 ± 0.05
TABLE 4
Backbone integration resulting from Russet Ranger transformation
IPT
PCR+ for
PCR+ for 0.6 kb
Binary vector
Total Nr.
phenotype
IPT
backbone fragment
pBI121
98
NA
NA
54 (55%)
pSIM108
193
138 (71%)
137 (71%)
NA
pSIM109
133
82 (62%)
80 (60%)
NA
NA: not applicable
TABLE 5
Backbone integration resulting from Russet Burbank transformation
Binary vector
Total Nr.
IPT phenotype
PSIM108
79
49 (60%)
PSIM109
72
60 (84%)
TABLE 6
Acrylamide levels in French fries derived from cold-stored
pSIM320 minitubers
Line
glucose mg/g (%-reduced)
acrylamide (PPB)
Untransformed
10.2
469
Vector control
10.2
NA
320-2
5.4 (47%)
95
320-4
5.8 (43%)
107
320-7
8.7 (14%)
353
320-9
7.4 (27%)
137
320-17
6.0 (41%)
506
320-21
8.5 (16%)
428
320-33
6.6 (35%)
516
NA: not available
TABLE 7
Acrylamide levels in French fries derived from
untransformed mature tubers
Stored at 18° C.
Stored at 4° C.
(color id.*)
(color id.*)
Glucose levels
<0.1 mg/g
3.4 mg/g
8-minute blanch
53 PPB (78)
603 PPB (56)
12-minute blanch
28 PPB (84)
244 PPB (71)
*: a higher value indicates a lighter color of the finished Fry product
TABLE 8
Glucose levels in cold-stored pSIM332 minitubers
Line
glucose mg/g (%-reduced)
Untransformed control
11.6 ± 0.5
Vector control
11.5 ± 0.5
332-1
5.4 (53%)
332-2
4.8 (58%)
332-4
7.0 (39%)
332-5
5.8 (50%)
332-6
6.9 (40%)
332-7
6.0 (48%)
332-8
6.8 (41%)
332-9
6.6 (43%)
332-10
5.4 (53%)
332-11
6.1 (47%)
332-12
6.4 (44%)
332-13
6.4 (44%)
332-15
7.7 (33%)
332-16
6.5 (43%)
332-17
5.3 (54%)
332-18
7.1 (38%)
332-21
6.3 (46%)
332-22
5.4 (53%)
332-23
4.2 (63%)
332-31
6.0 (48%)
332-34
6.2 (48%)
332-35
6.4 (44%)
332-39
6.7 (41%)
332-40
7.5 (35%)
332-41
5.7 (50%)
TABLE 9
Glucose levels in cold-stored pSIM216 minitubers
Line
glucose mg/g (%-reduced)
Untransformed control
11.6 ± 0.5
Vector control
11.5 ± 0.5
216-2
5.5 (52%)
216-3
8.8 (23%)
216-4
7.4 (36%)
216-5
5.8 (50%)
216-8
8.4 (27%)
216-10
5.1 (56%)
216-11
10.1 (19%)
216-12
9.3 (19%)
216-13
6.4 (44%)
216-15
8.8 (23%)
216-16
9.7 (16%)
216-17
6.4 (44%)
216-19
8.7 (24%)
216-21
3.2 (72%)
216-24
9.4 (18%)
216-26
9.3 (19%)
216-29
7.1 (38%)
216-30
8.2 (29%)
216-32
9.3 (19%)
216-34
7.1 (38%)
216-35
7.8 (32%)
216-38
7.1 (38%)
216-42
8.1 (30%)
216-44
9.4 (18%)
216-45
10.2 (11%)
TABLE 10
PPO activity in potato lines expressing a modified PPO gene
OD-410/gram
micro-tubers
mini-tubers
Line
(%-reduced)
(%-reduced)
Untransformed
24.59 ± 2.22
20.07 ± 1.21
controls
Vector controls
22.59 ± 3.36
19.55 ± 1.43
314-1
2.36
(90%)
17.8
(11%)
314-2
41.52
(−76%)
21.3
(−7%)
314-4
18.40
(22%)
5.4
(73%)
314-5
8.49
(64%)
19.1
(4%)
314-7
16.04
(32%)
16
(20%)
314-8
14.86
(37%)
17
(15%)
314-9
5.43
(77%)
4.3
(78%)
314-12
19.35
(18%)
19.6
(2%)
314-13
18.17
(23%)
15.4
(23%)
314-14
18.64
(21%)
17.32
(13%)
314-16
13.92
(41%)
18.2
(9%)
314-17
5.19
(78%)
2.4
(88%)
314-20
26.66
(−13%)
13.2
(34%)
314-21
11.32
(52%)
17.6
(12%)
314-22
13.45
(43%)
18.8
(6%)
314-23
5.19
(78%)
20.4
(−2%)
314-24
15.10
(36%)
19.6
(2%)
314-25
23.12
(2%)
19
(5%)
314-26
13.45
(43%)
17.8
(11%)
314-27
26.42
(−12%)
19.4
(3%)
314-28
31.85
(−35%)
19.4
(3%)
314-29
3.77
(84%)
14.8
(26%)
314-31
23.83
(−1%)
21.2
(−6%)
314-32
28.78
(−22%)
20
(0%)
TABLE 11
Table 11. PPO activity in potato minitubers expressing a modified
trailer sequence associated with the PPO gene
Line
OD-410/gram (%-reduced)
Untransformed controls
20.6 ± 1.3
Vector controls
17.9 ± 2.1
217-1
12.5 (39.4%)
217-4
12.6 (38.6%)
217-5
11.3 (45.0%)
217-6
6.1 (70.4%)
217-7
5.7 (72.5%)
217-9
10.4 (49.6%)
217-10
15.2 (26.3%)
217-11
15.2 (26.3%)
217-12
6.6 (67.9%)
217-14
15.4 (25.4%)
217-15
13.5 (34.6%)
217-16
6.0 (71.0%)
217-17
9.7 (53.0%)
217-19
8.6 (58.4%)
217-21
14.2 (31.1%)
217-22
9.7 (53.0%)
217-23
15.2 (26.3%)
217-24
8.2 (60.1%)
217-25
11.9 (42.2%)
217-26
3.1 (84.8%)
217-27
6.2 (69.9%)
217-29
7.2 (65.1%)
TABLE 12
Marker-free transformation with the LifeSupport vector + pSIM011
Gene-of-
Plant
Co-transformed
Marker only
interest only
Untransformed
Potato
0%
33%
11%
56%
Tobacco
20%
26%
18%
36%
Co-transformed: PCR-positive for both GUS and NPT
Gene-of-interest only: PCR-positive for GUS
Untransformed: Plants are PCR-negative for both GUS and NPT
TABLE 13
Sequential potato transformation with the LifeSupport vector and
pSIM011
Time
Gene-of-
window
Co-transformed
Marker only
interest only
Untransformed
0 hrs
9%
36%
9%
46%
4 hrs
20%
30%
20%
30%
Untransformed: Plants are PCR-negative for marker and gene-of-interest
TABLE 14
Marker-free transformation with the P-DNA vector pSIM340 +
LifeSupport
Gene-of-
Plant
Co-transformed
Marker only
interest only
Untransformed
Potato
17%
52.8%
1.2%
29%
Co-transformed: PCR-positive for both the PPO gene of pSIM340 and the
NPT gene from LifeSupport
Untransformed: Plants are PCR-negative for PPO and NPTII
TABLE 15
Marker-free potato transformation with pSIM340 + improved
LifeSupport vectors
LifeSupport
Co-
Gene-of-
vector
transformed
Marker only
interest only
Untransformed
PSIM346
0%
0%
4%
96%
PSIM350
10%
10%
29%
51%
PSIM401Ω
6%
34%
5%
55%
pSIM341Ω
16%
23%
7%
54%
Co-transformed: PCR-positive for both the PPO gene of pSIM340 and the
NPT gene from LifeSupport
Untransformed: Plants are PCR-negative for PPO and NPTII
TABLE 16
Marker-free potato transformation with a single Agrobacterium strain
carrying both pSIM356 and pSIM363
Gene-of-
Plant
Co-transformed
Marker only
interest only
Untransformed
Tobacco
50%
15%
19%
16%
Potato
22%
5%
15%
58%
Co-transformed: PCR-positive for both the GUS gene of pSIM356 and the
NPT gene from LifeSupport
Untransformed: Plants are PCR-negative for PPO and NPTII
SEQ ID NOs.
SEQ ID NO.: 1:
Potato P-DNA. Amplified fragment from
potato in Example 1.The bold underlined
portions of SEQ ID NO. 1 represent the left
(5′-) and right (3′-) border like sequences
termini of the P-DNA sequence
respectively
SEQ ID NO.: 2:
Wheat P-DNA Amplified fragment
from wheat in Example 1
SEQ ID NO.: 3:
Expression cassette for the IPT gene
SEQ ID NO.: 4:
Binary vectors pSIM111
SEQ ID NO.: 5:
Potato invertase inhibitor gene
SEQ ID NO.: 6:
Potato GBSS promoter
SEQ ID NO.: 7:
Potato Ubiquitin-3 gene terminator
SEQ ID NO.: 8:
Potato leader associated with the R1 gene
SEQ ID NO.: 9:
Potato Ubiquitin intron
SEQ ID NO.: 10:
Expression cassette for a sense
and antisense copy of the leader
associated with the R1 gene
SEQ ID NO.: 11:
Spacer
SEQ ID NO.: 12:
Alternative expression cassette
for a sense and antisense copy of the
leader associated with the R1 gene
SEQ ID NO.: 13:
Longer potato GBSS promoter
SEQ ID NO.: 14:
Alternative expression cassette
for a sense and antisense copy of the
leader associated with the R1 gene
SEQ ID NO.: 15:
Alternative expression cassette
for a sense and antisense copy of
the leader associated with the R1 gene
SEQ ID NO.: 16:
Potato trailer associated with the R1 gene
SEQ ID NO.: 17:
Expression cassette for a sense and
antisense copy of the trailer
associated with the R1 gene
SEQ ID NO.: 18:
Expression cassette for a sense and
antisense copy of the trailer
associated with the R1 gene
SEQ ID NO.: 19:
Expression cassette for a sense
nd antisense copy of the trailer
associated with the R1 gene
SEQ ID NO.: 20:
Expression cassette for a sense
and antisense copy of the trailer
associated with the R1 gene
SEQ ID NO.: 21:
Potato leader associated with the L
glucan phosphorylase gene
SEQ ID NO.: 22:
Potato trailer associated with the
L glucan phosphorylase gene
SEQ ID NO.: 23:
Expression cassette for a sense and
antisense copy of the leader
associated with the L glucan
phosphorylase gene
SEQ ID NO.: 24:
Alternative expression cassette
for a sense and antisense copy of
the leader associated with the L
glucan phosphorylase gene
SEQ ID NO.: 25:
Alternative expression cassette
for a sense and antisense copy of
the leader associated with the L
glucan phosphorylase gene
SEQ ID NO.: 26:
Alternative expression cassette for
a sense and antisense copy of the
leader associated with the L glucan
phosphorylase gene
SEQ ID NO.: 27:
Potato PPO gene
SEQ ID NO.: 28:
Modified inactive potato PPO gene
SEQ ID NO.: 29:
Potato trailer associated with a PPO gene
SEQ ID NO.: 30:
Expression cassette for a sense
and antisense copy of the
trailer associated with a PPO gene
SEQ ID NO.: 31:
Alternative expression cassette for
a sense and antisense copy of the
trailer associated with a PPO gene
SEQ ID NO.: 32:
Alternative expression cassette for
a sense and antisense copy of the
trailer associated with a PPO gene
SEQ ID NO.: 33:
Alternative expression cassette
for a sense andantisense copy of
the trailer associated with a PPO gene
SEQ ID NO.: 34:
Potato trailer associated with a
starch branching enzyme gene
SEQ ID NO.: 35:
Potato trailer associated with a
starch branching enzyme gene
SEQ ID NO.: 36:
Expression cassette for an
omega-mutated virD2 gene
SEQ ID NO.: 37:
Potato salt tolerance gene Pst1
SEQ ID NO.: 38:
Potato salt tolerance gene Pst2
SEQ ID NO.: 39:
Potato salt tolerance gene Pst3
SEQ ID NO.: 40:
Potato tuber specific promoter
SEQ ID NO.: 56:
Yeast ADH terminator
SEQ ID NO. 94:
Wheat left border-like sequence
SEQ ID NO. 95:
Wheat right border-like sequence
SEQ ID NO: 1
GTTTACAGTACCATATATCCTGTCAGAGGTATAGAGGCATGACTGGCATG
ATCACTAAATTGATGCCCACAGAGGAGACTTATAACCTACAGGGGCACGT
AGTTCTAGGACTTGAAAGTGACTGACCGTAGTCCAACTCGGTATAAAGCC
TACTCCCAACTAAATATATGAAATTTATAGCATAACTGCAGATGAGCTCG
ATTCTAGAGTAGGTACCGAGCTCGAATTCCTTACTCCTCCACAAAGCCGT
AACTGAAGCGACTTCTATTTTTCTCAACCTTCGGACCTGACGATCAAGAA
TCTCAATAGGTAGTTCTTCATAAGTGAGACTATCCTTCATAGCTACACTT
TCTAAAGGTACGATAGATTTTGGATCAACCACACACACTTCGTTTACACC
GGTATATATCCTGCCA
SEQ ID NO: 2
TGGCAGGATATATGAGTGTGTAAACAACCATAATCAGGCTGTAATTATC
AAGAGAACTAATGACAAGAAGCAGAGCTTATCAAGTGTTTCGTCCAGCTG
TAACATGGGCACAAAAGCTTGCTTGATGCATGTCTGGCTTTTCAAAGAGC
AATGTATTCTCAGGTACCTGCACGTTTGATCCCCTACCACGTACAAGACG
AGCAGAAAGGACATGTCTGCAGAAACTTAGACACATCCATTGCAGACTCG
TTCCGAAGCATCAGGAGAGTAGTCAGCAATGGTCATCTGCTGATGTAAAT
TAATTGATTGTTGGTAATCAAATTTTAACAGCAATATATATAATATATCA
ATAGTATATTGAACTATGAAAGACTGTAATCATATATAACAGCATACAAA
TTGTCGTGGAAACAAGAGGAGCTCATCAAGTGTTTAGTTCAGAAATAGCT
AACCAAGAATGCAATATAATAGGGGTACTGAGCTCCCTTCAAAATTACTA
ACTTCAGAAATAGCTAACCAAGAATGCAATGGCATTGCATAATTTAAACA
ACTGTCAGCACCAATCTCTGACTGAAGGCAGTTTACCCATTCAGAAGAGC
ACACATTTTCTGAACGACAACTCTGAGCGGGGATTGTTGACAGCAGCAAT
TAATCTGGCCTCAAGATGGTTTCCAACAACATAGATCAGATACAGCACTC
AAGCACCCAATAATCAGCCAGTACTGATCTGGTTACCACTGCAATTGATT
AACAGATGAACTGTGAAATTAAGATTTAACTGACAGTAATATATACCAGT
TGGCAGGATATATCCCTCTGTAAAC
SEQ ID NO: 3
CTGCAGCCAAAGCACATACTTATCGATTTAAATTTCATCGAAGAGATTAA
TATCGAATAATCATATACATACTTTAAATACATAACAAATTTTAAATACA
TATATCTGGTATATAATTAATTTTTTAAAGTCATGAAGTATGTATCAAAT
ACACATATGGAAAAAATTAACTATTCATAATTTAAAAAATAGAAAAGATA
CATCTAGTGAAATTAGGTGCATGTATCAAATACATTAGGAAAAGGGCATA
TATCTTGATCTAGATAATTAACGATTTTGATTTATGTATAATTTCCAAAT
GAAGGTTTATATCTACTTCAGAAATAACAATATACTTTTATCAGAACATT
CAACAAAGTAACAACCAACTAGAGTGAAAAATACACATTGTTCTCTAAAC
ATACAAAATTGAGAAAAGAATCTCAAAATTTAGAGAAACAAATCTGAATT
TCTAGAAGAAAAAAATAATTATGCACTTTGCTATTGCTCGAAAAATAAAT
GAAAGAAATTAGACTTTTTTAAAAGATGTTAGACTAGATATACTCAAAAG
CTATCAAAGGAGTAATATTCTTCTTACATTAAGTATTTTAGTTACAGTCC
TGTAATTAAAGACACATTTTAGATTGTATCTAAACTTAAATGTATCTAGA
ATACATATATTTGAATGCATCATATACATGTATCCGACACACCAATTCTC
ATAAAAAGCGTAATATCCTAAACTAATTTATCCTTCAAGTCAACTTAAGC
CCAATATACATTTTCATCTCTAAAGGCCCAAGTGGCACAAAATGTCAGGC
CCAATTACGAAGAAAAGGGCTTGTAAAACCCTAATAAAGTGGCACTGGCA
GAGCTTACACTCTCATTCCATCAACAAAGAAACCCTAAAAGCCGCAGCGC
CACTGATTTCTCTCCTCCAGGCGAAGATGCAGATCTTCGTGAAGACCCTA
ACGGGGAAGACGATCACCCTAGAGGTTGAGTCTTCCGACACCATCGACAA
TGTCAAAGCCAAGATCCAGGACAAGGAAGGGATTCCCCCAGACCAGCAGC
GTTTGATTTTCGCCGGAAAGCAGCTTGAGGATGGTCGTACTCTTGCCGAC
TACAACATCCAGAAGGAGTCAACTCTCCATCTCGTGCTCCGTCTCCGTGG
TGGTGGATCCATGGACCTGCATCTAATTTTCGGTCCAACTTGCACAGGAA
AGACGACGACCGCGATAGCTCTTGCCCAGCAGACAGGGCTTCCAGTCCTT
TCGCTTGATCGGGTCCAATGCTGTCCTCAACTATCAACCGGAAGCGGACG
ACCAACAGTGGAAGAACTGAAAGGAACGACGCGTCTCTACCTTGATGATC
GGCCTCTGGTGGAGGGTATCATCGCAGCCAAGCAAGCTCATCATAGGCTG
ATCGAGGAGGTGTATAATCATGAGGCCAACGGCGGGCTTATTCTTGAGGG
AGGATCCACCTCGTTGCTCAACTGCATGGCGCGAAACAGCTATTGGAGTG
CAGATTTTCGTTGGCATATTATTCGCCACAAGTTACCCGACCAAGAGACC
TTCATGAAAGCGGCCAAGGCCAGAGTTAAGCAGATGTTGCACCCCGCTGC
AGGCCATTCTATTATTCAAGAGTTGGTTTATCTTTGGAATGAACCTCGGC
TGAGGCCCATTCTGAAAGAGATCGATGGATATCGATATGCCATGTTGTTT
GCTAGCCAGAACCAGATCACGGCAGATATGCTATTGCAGCTTGACGCAAA
TATGGAAGGTAAGTTGATTAATGGGATCGCTCAGGAGTATTTCATCCATG
CGCGCCAACAGGAACAGAAATTCCCCCAAGTTAACGCAGCCGCTTTCGAC
GGATTCGAAGGTCATCCGTTCGGAATGTATTAGGTTACGCCAGCCCTGCG
TCGCACCTGTCTTCATCTGGATAAGATGTTCGTAATTGTTTTTGGCTTTG
TCCTGTTGTGGCAGGGCGGCAAATACTTCCGACAATCCATCGTGTCTTCA
AACTTTATGCTGGTGAACAAGTCTTAGTTTCCACGAAAGTATTATGTTAA
ATTTTAAAATTTCGATGTATAATGTGGCTATAATTGTAAAAATAAACTAT
CGTAAGTGTGAGTGTTATGTATAATTTGTCTAAATGTTTAATATATATCA
TAGAACGCAATAAATATTAAATATAGCGCTTTTATGAAATATAAATACAT
CATTACAAGTTGTTTATATTCGGGTGGACTAGTTTTTAATGTTTAGCAAA
TGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTT
GCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTG
ATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGA
TATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTC
AACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTC
TGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTT
ATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGGAATTC
SEQ ID NO: 4
AGCTTTGGCAGGATATATACCGGTGTAAACGAAGTGTGTGTGGTTGATCC
AAAATCTATCGTACCTTTAGAAAGTGTAGCTATGAAGGATAGTCTCACTT
ATGAAGAACTACCTATTGAGATTCTTGATCGTCAGGTCCGAAGGTTGAGA
AAAATAGAAGTCGCTTCAGTTACGGCTTTGTGGAGGAGTAAGGGTACCTA
CTCTAGAATCGAGCTCATCGTTATGCTATAAATTTCATATATTTAGTTGG
GAGTAGGCTTTATACCGAGTTGGACTACGGTCAGTCACTTTCAAGTCCTA
GAACTACGTGCCCCTGTAGGTTATAAGTCTCCTCTGTGGGCATCAATTTA
GTGATCATGCCAGTCATGCCTCTATACCTCTGACAGGATATATGGTACTG
TAAACACTAGTTGTGAATAAGTCGCTGTGTATGTTTGTTTGAGATCTCTA
AGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAA
GGTTTATCCGTTCGTCCATTTGTATGTGGTCACCTATCTCGAGCATGCCA
ACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCT
GCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAAC
GACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTT
TTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTT
GCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGC
CTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCA
ACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGA
TCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCAC
CTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCG
CAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCG
CGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCC
GGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTC
CCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAG
GCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCAC
GCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGC
ACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCG
AGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGAC
GCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGA
GGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCAT
TACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGC
CGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTG
TCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAAC
CGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGC
GTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGC
AAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCA
GGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGG
GGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATT
GGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGC
CCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGT
GATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGG
CAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGG
GCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGA
TGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGC
GCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATT
CTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGC
CGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGG
TCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAG
GTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCG
AGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCC
AGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGC
TGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCT
GAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTA
GATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCA
GGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCC
AGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCC
CCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACA
AATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGC
AGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCG
TGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGC
AGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAG
ATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGC
ATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGG
CGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAG
GGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCG
GTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGA
CAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCT
GCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGC
ATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAA
GAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCT
ACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAG
CTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGT
GCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTT
TTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGG
TTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAA
GTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGT
ACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGC
TACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGA
GCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTC
TCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGG
AACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTC
ACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCT
AAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCA
TAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCT
TCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGG
CCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGG
CGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGG
CACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATG
CAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAG
ACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAG
CCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATG
CGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATA
CCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTC
CTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTAT
CAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAAC
GCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAA
AAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGC
ATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTA
TAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGT
TCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAA
GCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAG
GTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGA
CCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC
ACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCG
AGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG
CTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTA
CCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCT
GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAA
AGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT
GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTAGGTACTAA
AACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTT
GATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATAT
CCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCC
CTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCAC
AAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTT
CGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAAT
GGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATT
CAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGG
GACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATAC
AGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCA
AAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCC
GTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGC
ATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCT
GTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATA
CCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCG
ATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATTTATTATTT
CCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGCCGCTAATTATAA
CAAGACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACC
AGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTATAACATAGTA
TCGACGGAGCCGATTTTGAAACCGCGGATCCTGCAGCCAAAGCACATACT
TATCGATTTAAATTTCATCGAAGAGATTAATATCGAATAATCATATACAT
ACTTTAAATACATAACAAATTTTAAATACATATATCTGGTATATAATTAA
TTTTTTAAAGTCATGAAGTATGTATCAAATACACATATGGAAAAAATTAA
CTATTCATAATTTAAAAAATAGAAAAGATACATCTAGTGAAATTAGGTGC
ATGTATCAAATACATTAGGAAAAGGGCATATATCTTGATCTAGATAATTA
ACGATTTTGATTTATGTATAATTTCCAAATGAAGGTTTATATCTACTTCA
GAAATAACAATATACTTTTATCAGAACATTCAACAAAGTAACAACCAACT
AGAGTGAAAAATACACATTGTTCTCTAAACATACAAAATTGAGAAAAGAA
TCTCAAAATTTAGAGAAACAAATCTGAATTTCTAGAAGAAAAAAATAATT
ATGCACTTTGCTATTGCTCGAAAAATAAATGAAAGAAATTAGACTTTTTT
AAAAGATGTTAGACTAGATATACTCAAAAGCTATCAAAGGAGTAATATTC
TTCTTACATTAAGTATTTTAGTTACAGTCCTGTAATTAAAGACACATTTT
AGATTGTATCTAAACTTAAATGTATCTAGAATACATATATTTGAATGCAT
CATATACATGTATCCGACACACCAATTCTCATAAAAAGCGTAATATCCTA
AACTAATTTATCCTTCAAGTCAACTTAAGCCCAATATACATTTTCATCTC
TAAAGGCCCAAGTGGCACAAAATGTCAGGCCCAATTACGAAGAAAAGGGC
TTGTAAAACCCTAATAAAGTGGCACTGGCAGAGCTTACACTCTCATTCCA
TCAACAAAGAAACCCTAAAAGCCGCAGCGCCACTGATTTCTCTCCTCCAG
GCGAAGATGCAGATCTTCGTGAAGACCCTAACGGGGAAGACGATCACCCT
AGAGGTTGAGTCTTCCGACACCATCGACAATGTCAAAGCCAAGATCCAGG
ACAAGGAAGGGATTCCCCCAGACCAGCAGCGTTTGATTTTCGCCGGAAAG
CAGCTTGAGGATGGTCGTACTCTTGCCGACTACAACATCCAGAAGGAGTC
AACTCTCCATCTCGTGCTCCGTCTCCGTGGTGGTGGATCCATGGACCTGC
ATCTAATTTTCGGTCCAACTTGCACAGGAAAGACGACGACCGCGATAGCT
CTTGCCCAGCAGACAGGGCTTCCAGTCCTTTCGCTTGATCGGGTCCAATG
CTGTCCTCAACTATCAACCGGAAGCGGACGACCAACAGTGGAAGAACTGA
AAGGAACGACGCGTCTCTACCTTGATGATCGGCCTCTGGTGGAGGGTATC
ATCGCAGCCAAGCAAGCTCATCATAGGCTGATCGAGGAGGTGTATAATCA
TGAGGCCAACGGCGGGCTTATTCTTGAGGGAGGATCCACCTCGTTGCTCA
ACTGCATGGCGCGAAACAGCTATTGGAGTGCAGATTTTCGTTGGCATATT
ATTCGCCACAAGTTACCCGACCAAGAGACCTTCATGAAAGCGGCCAAGGC
CAGAGTTAAGCAGATGTTGCACCCCGCTGCAGGCCATTCTATTATTCAAG
AGTTGGTTTATCTTTGGAATGAACCTCGGCTGAGGCCCATTCTGAAAGAG
ATCGATGGATATCGATATGCCATGTTGTTTGCTAGCCAGAACCAGATCAC
GGCAGATATGCTATTGCAGCTTGACGCAAATATGGAAGGTAAGTTGATTA
ATGGGATCGCTCAGGAGTATTTCATCCATGCGCGCCAACAGGAACAGAAA
TTCCCCCAAGTTAACGCAGCCGCTTTCGACGGATTCGAAGGTCATCCGTT
CGGAATGTATTAGGTTACGCCAGCCCTGCGTCGCACCTGTCTTCATCTGG
ATAAGATGTTCGTAATTGTTTTTGGCTTTGTCCTGTTGTGGCAGGGCGGC
AAATACTTCCGACAATCCATCGTGTCTTCAAACTTTATGCTGGTGAACAA
GTCTTAGTTTCCACGAAAGTATTATGTTAAATTTTAAAATTTCGATGTAT
AATGTGGCTATAATTGTAAAAATAAACTATCGTAAGTGTGCGTGTTATGT
ATAATTTGTCTAAATGTTTAATATATATCATAGAACGCAATAAATATTAA
ATATAGCGCTTTTATGAAATATAAATACATCATTACAAGTTGTTTATATT
TCGGGTGGACTAGTTTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCT
TTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTT
TTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGA
GTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATA
TCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGC
TGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATG
CTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTA
TAATATAGTAAAAAAATAGGAATTCTATCCGCGGTGATCACAGGCAGCAA
CGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGT
GTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAAC
ATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCG
GACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGT
CGGGGAGCTGTTGGCTGGCTGGA
SEQ ID NO: 5
ATGAGAAATTTATTCCCCATATTGATGCTAATCACCAATTTGGCACTCAA
CAACGATAACAACAACAACAACAACAACAACAATAATTATAATCTCATAC
ACGCAACGTGTAGGGAGACCCCATATTACTCCCTATGTCTCACCACCCTA
CAATCCGGTCCACGTAGTAACGAGGTTGAGGGTGGTGATGCCATCACCAC
CCTAGGCCTCATCATGGTGGACGCGGTGAAATCAAAGTCCATAGAAATAA
TGGAAAAAATAAAAGAGCTAGAGAAATCGAACCCTGAGTGGCGGGCCCCA
CTTAGCCAGTGTTACGTGGCGTATAATGCCGTCCTACGAGCCGATGTAAC
GGTAGCCGTTGAAGCCTTAAAGAAGGGTGCCCCCAAATTTGCTGAAGATG
GTATGGATGATGTTGTTGCTGAAGCACAAACTTGTGAGTATAGTTTTAAT
TATTATAATAAATTGGATTTTCCAATTTCTAATTTGAGTAGGGAAATAAT
TGAACTATCAAAAGTTGCTAAATCCATAATTAGAATGTTATTATGA
SEQ ID NO: 6
GAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTG
GAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAAT
ATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGG
AGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATT
GCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCAT
AATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTC
CGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCT
GCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACAC
AGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTT
CATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAAC
GTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTAC
TGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCT
TCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAG
CTTGGTAG
SEQ ID NO: 7
TTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGT
AATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGAC
AACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTG
TTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCT
CTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGAT
TATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTT
GAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAA
AATAG
SEQ ID NO: 8
ACCTTATTTCACTACCACTTTCCACTCTCCAATCCCCATACTCTCTGCTC
CAATCTTCATTTTGCTTCGTGAATTCATCTTCATCGAATTTCTCGACGCT
TCTTCGCTAATTTCCTCGTTACTTCACTAAAAATCGACGTTTCTAGCTGA
ACTTGAGTGAATTAAGCCAGTGGGAGGAT
SEQ ID NO: 9
GTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATT
AGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTG
TTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGG
AAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCT
AGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGA
GTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGT
TGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTC
TGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTA
AATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTT
TTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCA
GGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGT
TGTTTTTATGAATTTGCAG
SEQ ID NO: 10
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAAT
TCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTA
TAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGT
AGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGG
GCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAG
GGCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGT
TGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACT
GAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCA
CTCACACAGCTCAAGAAGGATCCACCTTATTTCACTACCACTTTCCACTC
TCCAATCCCCATACTCTCTGCTCCAATCTTCATTTTGCTTCGTGAATTCA
TCTTCATCGAATTTCTCGACGCTTCTTCGCTAATTTCCTCGTTACTTCAC
TAGAAATCGACGTTTCTAGCTGAACTTGAGTGAATTAAGCCAGTGGGAGG
ATGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAG
ATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTT
TTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGT
TGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATG
AAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGT
TTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGA
GTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGT
CGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATG
AAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTA
AATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTCCTTCTCGAA
TCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTT
GTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCATCCTCCCACT
GGCTTAATTCACTCAAGTTCAGCTAGAAACGTCGATTTCTAGTGAAGTAA
CGAGGAAATTAGCGAAGAAGCGTCGAGAAATTCGATGAAGATGAATTCAC
GAAGCAAAATGAAGATTGGAGCAGAGAGTATGGGGATTGGAGAGTGGAAA
GTGGTAGTGAAATAAGGTAAGCTTTTGATTTTAATGTTTAGCAAATGTCC
TATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTAT
ATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTA
TTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCA
ATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACAC
ACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGG
GATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCT
CATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA
SEQ ID NO: 11
GTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCC
CTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATC
TCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCT
ACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTC
AGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAG
SEQ ID NO: 12
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAAGAAGGATCCACCTTATTTCACTACCACTTTCCACTCT
CCAATCCCCATACTCTCTGCTCCAATCTTCATTTTGCTTCGTGAATTCAT
CTTCATCGAATTTCTCGACGCTTCTTCGCTAATTTCCTCGTTACTTCACT
AGAAATCGACGTTTCTAGCTGAACTTGAGTGAATTAAGCCAGTGGGAGGA
TGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCAT
GCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTT
GTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGA
ATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCT
TCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTT
GATATCATCCTCCCACTGGCTTAATTCACTCAAGTTCAGCTAGAAACGTC
GATTTCTAGTGAAGTAACGAGGAAATTAGCGAAGAAGCGTCGAGAAATTC
GATGAAGATGAATTCACGAAGCAAAATGAAGATTGGAGCAGAGAGTATGG
GGATTGGAGAGTGGAAAGTGGTAGTGAAATAAGGTAAGCTTTTGATTTTA
ATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTT
AGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCC
TCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTC
GTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACG
TGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGG
AACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGAT
TTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAG
TCTAGA
SEQ ID NO: 13
GAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTG
GAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAAT
ATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGG
AGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATT
GCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCAT
AATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTC
CGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCT
GCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACAC
AGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTT
CATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAAC
GTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTAC
TGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCT
TCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAG
CTTGGTAGATTCCCCTTTTTGTAGACCACACATCAC
SEQ ID NO: 14
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAAGAAGGATCCTCATATTCTAGTTGTATGTTGTTCAGAG
AAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTT
ACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGC
CATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGA
ATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCT
GGGTATGATATAGGGAGAAATGTGTAAACATTGTACTATATATAGTATAT
ACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAA
AGAAGGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTT
TAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAG
TTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTA
CGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTG
ATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAA
GGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATT
TGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTT
CGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTG
ATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCC
CTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTC
GAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTC
GTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTCTTTG
ATGCTGCGGTATTATTCAGTGCATAATGCAATACATAATGCGTGTGTATA
TACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATACCCAGC
TAATTTAGCTTTTACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCC
TACTTCTCACTTCTCCTCCAATAGCTGAAGGTGAACATGATGTGATGGCT
TTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAAG
TGGTCACAGATCTGATACAATGAGAATATGATCACATCTGTGGTCTTCTC
TGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTAGCA
AATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTT
TTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGT
TGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTG
GATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGT
TCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAG
TCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCT
TTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA
SEQ ID NO: 15
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAAGAAGGATCCTCATATTCTAGTTGTATGTTGTTCAGAG
AAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTT
ACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGC
CATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGA
ATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCT
GGGTATGATATAGGGAGAAATGTGTAAACATTGTACTATATATAGTATAT
ACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAA
AGAAGGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATT
TCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAA
CGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTA
CTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTC
TTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTA
GCTTGATATCCTTCTTTGATGCTGCGGTATTATTCAGTGCATAATGCAAT
ACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCT
CCCTATATCATACCCAGCTAATTTAGCTTTTACAAAGTTTTTCTTATTAT
TCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAAGGT
GAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCA
TGGGAGGTATCAGGTAAGTGGTCACAGATCTGATACAATGAGAATATGAT
CACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTT
TGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAAC
GGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGT
GACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTA
TTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGT
TCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATG
GATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAA
TTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAA
AAAAATAGTCTAGA
SEQ ID NO: 16
TCATATTCTAGTTGTATGTTGTTCAGAGAAGACCACAGATGTGATCATAT
TCTCATTGTATCAGATCTGTGACCACTTACCTGATACCTCCCATGAAGTT
ACCTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCA
GCTATTGGAGGAGAAGTGAGAAGTAGGAATTGCAATATGAGGAATAATAA
GAAAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATAGGGAGAAATG
TGTAAACATTGTACTATATATAGTATATACACACGCATTATGTATTGCAT
TATGCACTGAATAATACCGCAGCATCAAAGAAG
SEQ ID NO: 17
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAAGAAGGATCCTCATATTCTAGTTGTATGTTGTTCAGAG
AAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTT
ACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGC
CATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGA
ATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCT
GGGTATGATATAGGGAGAAATGTGTAAACATTGTACTATATATAGTATAT
ACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAA
AGAAGGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTT
TAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAG
TTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTA
CGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTG
ATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAA
GGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATT
TGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTT
CGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTG
ATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCC
CTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTC
GAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTC
GTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTCTTTG
ATGCTGCGGTATTATTCAGTGCATAATGCAATACATAATGCGTGTGTATA
TACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATACCCAGC
TAATTTAGCTTTTACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCC
TACTTCTCACTTCTCCTCCAATAGCTGAAGGTGAACATGATGTGATGGCT
TTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAAG
TGGTCACAGATCTGATACAATGAGAATATGATCACATCTGTGGTCTTCTC
TGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTAGCA
AATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTT
TTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGT
TGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTG
GATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGT
TCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAG
TCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCT
TTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA
SEQ ID NO: 18
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAAGAAGGATCCTCATATTCTAGTTGTATGTTGTTCAGAG
AAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTT
ACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGC
CATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGA
ATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCT
GGGTATGATATAGGGAGAAATGTGTAAACATTGTACTATATATAGTATAT
ACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAA
AGAAGGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATT
TCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAA
CGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTA
CTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTC
TTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTA
GCTTGATATCCTTCTTTGATGCTGCGGTATTATTCAGTGCATAATGCAAT
ACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCT
CCCTATATCATACCCAGCTAATTTAGCTTTTACAAAGTTTTTCTTATTAT
TCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAAGGT
GAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCA
TGGGAGGTATCAGGTAAGTGGTCACAGATCTGATACAATGAGAATATGAT
CACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTT
TGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAAC
GGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGT
GACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTA
TTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGT
TCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATG
GATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAA
TTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAA
AAAAATAGTCTAGA
SEQ ID NO: 19
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTC
TGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGT
ATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTT
GCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCC
TCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCAT
CTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACATCACGGATCCTC
ATATTCTAGTTGTATGTTGTTCAGAGAAGACCACAGATGTGATCATATTC
TCATTGTATCAGATCTGTGACCACTTACCTGATACCTCCCATGAAGTTAC
CTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCAGC
TATTGGAGGAGAAGTGAGAAGTAGGAATTGCAATATGAGGAATAATAAGA
AAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATAGGGAGAAATGTG
TAAACATTGTACTATATATAGTATATACACACGCATTATGTATTGCATTA
TGCACTGAATAATACCGCAGCATCAAAGAAGGAATTCAAGGTTAGAAATC
TTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAG
GTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGGA
AAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTA
CAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGAT
TTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACAT
TTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATG
AAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTA
AAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTT
TCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTG
TTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGA
AAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATG
AATTTGCAGATGGATATCCTTCTTTGATGCTGCGGTATTATTCAGTGCAT
AATGCAATACATAATGCGTGTGTATATACTATATATAGTACAATGTTTAC
ACATTTCTCCCTATATCATACCCAGCTAATTTAGCTTTTACAAAGTTTTT
CTTATTATTCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAG
CTGAAGGTGAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGG
TAACTTCATGGGAGGTATCAGGTAAGTGGTCACAGATCTGATACAATGAG
AATATGATCACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATG
AAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTT
TGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTG
ATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTT
TTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCT
TTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGC
AGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTT
CTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAA
TATAGTAAAAAAATAGTCTAGA
SEQ ID NO: 20
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAAGAAGGATCCTCATATTCTAGTTGTATGTTGTTCAGAG
AAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTT
ACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGC
CATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGA
ATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCT
GGGTATGATATAGGGAGAAATGTGTAAACATTGTACTATATATAGTATAT
ACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAA
AGAAGGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATT
TCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAA
CGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTA
CTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTC
TTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTA
GCTTGATATCCTTCTTTGATGCTGCGGTATTATTCAGTGCATAATGCAAT
ACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCT
CCCTATATCATACCCAGCTAATTTAGCTTTTACAAAGTTTTTCTTATTAT
TCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAAGGT
GAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCA
TGGGAGGTATCAGGTAAGTGGTCACAGATCTGATACAATGAGAATATGAT
CACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTT
TGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAAC
GGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGT
GACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTA
TTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGT
TCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATG
GATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAA
TTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAA
AAAAATAGTCTAGA
SEQ ID NO: 21
TTAGAGTGTGGGTAAGTAATTAAGTTAGGGATTTGTGGGAAATGGACAAA
TATAAGAGAGTGCAGGGGAGTAGTGCAGGAGATTTTCGTGCTTTTATTGA
TAAATAAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACAAC
ACACTTAATTCCTGTGTGTGAATCAATAATTGACTTCTCCAATCTTCATC
AATAAAATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGC
TAGATTTGCATAGAGAGCACAAA
SEQ ID NO: 22
GAGGGGGAAGTGAATGAAAAATAACAAAGGCACAGTAAGTAGTTTCTCTT
TTTATCATGTGATGAAGGTATATAATGTATGTGTAAGAGGATGATGTTAT
TACCACATAATAAGAGATGAAGAGTCTCATTTTCTGCTTAAAAAAACAAT
TCACTGGC
SEQ ID NO: 23
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAAGAAGGATCCGAGTGTGGGTAAGTAATTAAGTTAGGGA
TTTGTGGGAAATGGACAAATATAAGAGAGTGCAGGGGAGTAGTGCAGGAG
ATTTTCGTGCTTTTATTGATAAATAAAAAAAGGGTGACATTTAATTTCCA
CAAGAGGACGCAACACAACACACTTAATTCCTGTGTGTGAATCAATAATT
GACTTCTCCAATCTTCATCAATAAAATAATTCACAATCCTCACTCTCTTA
TCACTCTCATTCGAAAAGCTAGATTTGCATAGAGAGCACAGAATTCAAGG
TTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATTA
GCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTGT
TTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGA
AAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTA
GTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAG
TTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTT
GATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCT
GAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAA
ATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTT
TTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAG
GGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTT
GTTTTTATGAATTTGCAGATGGATATCTGTGCTCTCTATGCAAATCTAGC
TTTTCGAATGAGAGTGATAAGAGAGTGAGGATTGTGAATTATTTTATTGA
TGAAGATTGGAGAAGTCAATTATTGATTCACACACAGGAATTAAGTGTGT
TGTGTTGCGTCCTCTTGTGGAAATTAAATGTCACCCTTTTTTTATTTATC
AATAAAAGCACGAAAATCTCCTGCACTACTCCCCTGCACTCTCTTATATT
TGTCCATTTCCCACAAATCCCTAACTTAATTACTTACCCACACTCTAAGC
TTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCG
AACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTA
TGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGC
TTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCT
AGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGT
ATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAG
GAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAG
TAAAAAAATAGTCTAGA
SEQ ID NO: 24
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAAGAAGGATCCGAGTGTGGGTAAGTAATTAAGTTAGGGA
TTTGTGGGAAATGGACAAATATAAGAGAGTGCAGGGGAGTAGTGCAGGAG
ATTTTCGTGCTTTTATTGATAAATAAAAAAAGGGTGACATTTAATTTCCA
CAAGAGGACGCAACACAACACACTTAATTCCTGTGTGTGAATCAATAATT
GACTTCTCCAATCTTCATCAATAAAATAATTCACAATCCTCACTCTCTTA
TCACTCTCATTCGAAAAGCTAGATTTGCATAGAGAGCACAGAATTCGTGG
TAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCC
TACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCT
CATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTA
CTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCA
GAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCTGTGC
TCTCTATGCAAATCTAGCTTTTCGAATGAGAGTGATAAGAGAGTGAGGAT
TGTGAATTATTTTATTGATGAAGATTGGAGAAGTCAATTATTGATTCACA
CACAGGAATTCCGTGTGTTGTGTTGCGTCCTCTTGTGGAAATTAAATGTC
ACCCTTTTTTTATTTATCAATAAAAGCACGAAAATCTCCTGCACTACTCC
CCTGCACTCTCTTATATTTGTCCATTTCCCACAAATCCCTAACTTAATTA
CTTACCCACACTCTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATC
AGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGG
ATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTC
AAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCT
TAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTA
GCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATC
GATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATT
GGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA
SEQ ID NO: 25
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTC
TGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGT
ATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTT
GCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCC
TCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCAT
CTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACATCACGGATCCGA
GTGTGGGTAAGTAATTAAGTTAGGGATTTGTGGGAAATGGACAAATATAA
GAGAGTGCAGGGGAGTAGTGCAGGAGATTTTCGTGCTTTTATTGATAAAT
AAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACAACACACT
TAATTCCTGTGTGTGAATCAATAATTGACTTCTCCAATCTTCATCAATAA
AATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGCTAGAT
TTGCATAGAGAGCACAGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGT
TTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCC
TTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTT
GAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCC
CCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGAT
TCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAA
GGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAAT
TTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTC
CGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTG
ATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGG
TTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTT
GAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGAT
ATCTGTGCTCTCTATGCAAATCTAGCTTTTCGAATGAGAGTGATAAGAGA
GTGAGGATTGTGAATTATTTTATTGATGAAGATTGGAGAAGTCAATTATT
GATTCACACACAGGAATTAAGTGTGTTGTGTTGCGTCCTCTTGTGGAAAT
TAAATGTCACCCTTTTTTTATTTATCAATAAAAGCACGAAAATCTCCTGC
ACTACTCCCCTGCACTCTCTTATATTTGTCCATTTCCCACAAATCCCTAA
CTTAATTACTTACCCACACTCTAAGCTTTTGATTTTAATGTTTAGCAAAT
GTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTG
CTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGA
TTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGAT
ATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCA
ACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCT
GTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTA
TGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA
SEQ ID NO: 26
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTC
TGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGT
ATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTT
GCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCC
TCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCAT
CTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACATCACGGATCCGA
GTGTGGGTAAGTAATTAAGTTAGGGATTTGTGGGAAATGGACAAATATAA
GAGAGTGCAGGGGAGTAGTGCAGGAGATTTTCGTGCTTTTATTGATAAAT
AAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACAACACACT
TAATTCCTGTGTGTGAATCAATAATTGACTTCTCCAATCTTCATCAATAA
AATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGCTAGAT
TTGCATAGAGAGCACAGAATTCGTGGTAACTTTTACTCATCTCCTCCAAT
TATTTATGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTA
TGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGAT
TCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAAT
GCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTT
GTTCATCTGTAGCTTGATATCTGTGCTCTCTATGCAAATCTAGCTTTTCG
AATGAGAGTGATAAGAGAGTGAGGATTGTGAATTATTTTATTGATGAAGA
TTGGAGAAGTCAATTATTGATTCACACACAGGAATTAAGTGTGTTGTGTT
GCGTCCTCTTGTGGAAATTAAATGTCACCCTTTTTTTATTTATCAATAAA
AGCACGAAAATCTCCTGCACTACTCCCCTGCACTCTCTTATATTTGTCCA
TTTCCCACAAATCCCTAACTTAATTACTTACCCACACTCTAAGCTTTTGA
TTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGT
AATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGAC
AACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTG
TTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCT
CTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGAT
TATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTT
GAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAA
AATAGTCTAGA
SEQ ID NO: 27
ATGGCAAGCTTGTGCAATAGTAGTAGTACATCTCTCAAAACTCCTTTTAC
TTCTTCCTCCACTTCTTTATCTTCCACTCCTAAGCCCTCTCAACTTTTCA
TCCATGGAAAACGTAACCAAATGTTCAAAGTTTCATGCAAGGTTATCAAT
AATAACGGTGACCAAAACGTTGAAACGAATTCTGTTGATCGAAGAAATGT
TCTTCTTGGCTTAGGTGGTCTTTATGGTGTTGCTAATGCTATACCATTAG
CTGCATCCGCTGCTCCAACTCCACCTCCTGATCTCTCGTCTTGTAGTATA
GCCAGGATTAACGAAAATCAGGTGGTGCCGTACAGTTGTTGCGCGCCTAA
GCCTGATGATATGGAGAAAGTTCCGTATTACAAGTTCCCTTCTATGACTA
AGCTCCGTGTCCGTCAGCCTGCTCATGAAGCTAATGAGGAGTATATTGCC
AAGTACAATCTGGCGATTAGTCGAATGAGAGATCTTGATAAGACACAACC
TTTAAACCCTATTGGTTTTAAGCAACAAGCTAATATACATTGTGCTTATT
GTAATGGTGCTTATAGAATTGGTGGCAAAGAGTTACAAGTTCATAATTCT
TGGCTTTTCTTCCCGTTCCATAGATGGTACTTGTACTTCCACGAGAGAAT
CGTGGGAAAATTCATTGATGATCCAACTTTCGCTTTGCCATATTGGAATT
GGGACCATCCAAAGGGTATGCGTTTTCCTGCCATGTATGATCGTGAAGGG
ACTTCCCTTTTCGATGTAACACGTGACCAAAGTCACCGAAATGGAGCAGT
AATCGATCTTGGTTTTTTCGGCAATGAAGTCGAAACAACTCAACTCCAGT
TGATGAGCAATAATTTAACACTAATGTACCGTCAAATGGTAACTAATGCT
CCATGTCCTCGGATGTTCTTTGGTGGGCCTTATGATCTCGGGATTAACAC
TGAACTCCCGGGAACTATAGAAAACATTCCTCACGGTCCTGTCCACATCT
GGTCTGGTACAGTGAGAGGTTCAACTTTGCCCAATGGTGCAATATCAAAC
GGTGAGAATATGGGTCATTTTTACTCAGCTGCTTTGGACCCGGTTTTCTT
TTGCCATCACAGCAATGTGGATCGGATGTGGAGCGAATGGAAAGCGACAG
GAGGGAAAAGAACAGATATCACACATAAAGGTTGGTTGAACTCCGAGTTC
TTTTTCTATGATGAAAATGAAAACCCTTACCGTGTGAAAGTCCGAGACTG
TTTGGACACGAAGAAGATGGGGTATGATTATGCACCAATGGCCACCCCGT
GGCGTAACTTCAAGCCAATAACAAAAACTACAGCTGGGAAAGTGAATACA
GCTTCTCTTCCGCCAGCTAGCAATGTATTCCCAGTGGCTAAACTCGACAA
AGCAATTTCGTTTTCCATCAATAGGCCGACTTCGTCAAGGACTCAACAAG
AGAAAAATGCACAAGAGGAGATGTTGACATTCAGTAGCATAAGATATGAT
AACAGAGGGTACATAAGGTTCGATGTGTTCCTGAACGTGGACAATAATGT
GAATGCGAATGAGCTTGACAAGGCGGAGTTTGCGGGGAGTTATACTAGTT
TGCCACATGTTCATAGAGCTGGTGAGACTAATCATATCGCGACTGTTGAT
TTCCAGCTGGCGATAACGGAACTGTTGGAGGATATTGGTTTGGAAGATGA
AGATACTATTGCGGTGACTCTGGTGCCAAAGAGAGGTGGTGAAGGTATCT
CCATTGAAAGTGCGACGATCAGTCTTGCAGATTGTTAA
SEQ ID NO: 28
ATGGCAAGCTTGTGCAATAGTAGTAGTACATCTCTCAAAACTCCTTTTAC
TTCTTCCTCCACTTCTTTATCTTCCACTCCTAAGCCCTCTCAACTTTTCA
TCCATGGAAAACGTAACCAAATGTTCAAAGTTTCATGCAAGGTTATCAAT
AATAACGGTGACCAAAACGTTGAAACGAATTCTGTTGATCGAAGAAATGT
TCTTCTTGGCTTAGGTGGTCTTTATGGTGTTGCTAATGCTATACCATTAG
CTGCATCCGCTGCTCCAACTCCACCTCCTGATCTCTCGTCTTGTAGTATA
GCCAGGATTAACGAAAATCAGGTGGTGCCGTACAGTTGTTGCGCGCCTAA
GCCTGATGATATGGAGAAAGTTCCGTATTACAAGTTCCCTTCTATGACTA
AGCTCCGTGTCCGTCAGCCTGCTCATGAAGCTAATGAGGAGTATATTGCC
AAGTACAATCTGGCGATTAGTCGAATGAGAGATCTTGATAAGACACAACC
TTTAAACCCTATTGGTTTTAAGCAACAAGCTAATATACAGTGGGCTTATG
GTAATGGTGCTTATAGAATTGGTGGCAAAGAGTTACAAGTTCATAATTCT
TGGCTTTTCTTCCCGTTCCATAGATGGTACTTGTACTTCCACGAGAGAAT
CGTGGGAAAATTCATTGATGATCCAACTTTCGCTTTGCCATATTGGAATT
GGGACCATCCAAAGGGTATGCGTTTTCCTGCCATGTATGATCGTGAAGGG
ACTTCCCTTTTCGATGTAACACGTGACCAAAGTCACCGAAATGGAGCAGT
AATCGATCTTGGTTTTTTCGGCAATGAAGTCGAAACAACTCAACTCCAGT
TGATGAGCAATAATTTAACACTAATGTACCGTCAAATGGTAACTAATGCT
CCATGTCCTCGGATGTTCTTTGGTGGGCCTTATGATCTCGGGATTAACAC
TGAACTCCCGGGAACTATAGGAAACATTCCTCTCGGTCCTGTCCACATCT
GGTCTGGTACAGTGAGAGGTTCAACTTTGCCCAATGGTGCAATATCAAAC
GGTGAGAATATGGGTCATTTTTACTCAGCTGCTTTGGACCCGGTTTTCTT
TTGCCATCACAGCAATGTGGATCGGATGTGGAGCGAATGGAAAGCGACAG
GAGGGAAAAGAACAGATATCACACATAAAGGTTGGTTGAACTCCGAGTTC
TTTTTCTATGATGAAAATGAAAACCCTTACCGTGTGAAAGTCCGAGACTG
TTTGGACACGAAGAAGATGGGGTATGATTATGCACCAATGGCCACCCCGT
GGCGTAACTTCAAGCCAATAACAAAAACTACAGCTGGGAAAGTGAATACA
GCTTCTCTTCCGCCAGCTAGCAATGTATTCCCAGTGGCTAAACTCGACAA
AGCAATTTCGTTTTCCATCAATAGGCCGACTTCGTCAAGGACTCAACAAG
AGAAAAATGCACAAGAGGAGATGTTGACATTCAGTAGCATAAGATATGAT
AACAGAGGGTACATAAGGTTCGATGTGTTCCTGAACGTGGACAATAATGT
GAATGCGAATGAGCTTGACAAGGCGGAGTTTGCGGGGAGTTATACTAGTT
TGCCACATGTTCATAGAGCTGGTGAGACTAATCATATCGCGACTGTTGAT
TTCCAGCTGGCGATAACGGAACTGTTGGAGGATATTGGTTTGGAAGATGA
AGATACTATTGCGGTGACTCTGGTGCCAAAGAGAGGTGGTGAAGGTATCT
5CCATTGAAAGTGCGACGATCAGTCTTGCAGATTGTTAA
SEQ ID NO: 29
TTAGTCTCTATTGAATCTGCTGAGATTACACTTTGATGGATGATGCTCTG
TTTTTGTTTTCTTGTTCTGTTTTTTCCTCTGTTGAAATCAGCTTTGTTGC
TTGATTTCATTGAAGTTGTTATTCAAGAATAAATCAGTTACAATTATGTT
TGGG
SEQ ID NO: 30
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAAGAAGGATCCTTAGTCTCTATTGAATCTGCTGAGATTA
CACTTTGATGGATGATGCTCTGTTTTTGTTTTCTTGTTCTGTTTTTTCCT
CTGTTGAAATCAGCTTTGTTGCTTGATTTCATTGAAGTTGTTATTCAAGA
ATAAATCAGTTACAATTATGGAATTCAAGGTTAGAAATCTTCTCTATTTT
TGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATA
GCCCTTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAA
ATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTT
TTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGT
CGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGA
AAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTA
GAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTT
TCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGT
TTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGT
TTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAA
ATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGAT
GGATATCCTTCTTTGATGCTGATCCATAATTGTAACTGATTTATTCTTGA
ATAACAACTTCAATGAAATCAAGCAACAAAGCTGATTTCAACAGAGGAAA
AAACAGAACAAGAAAACAAAAACAGAGCATCATCCATCAAAGTGTAATCT
CAGCAGATTCAATAGAGACTAAGCTTTTGATTTTAATGTTTAGCAAATGT
CCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCT
ATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATT
TATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATAT
CAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAAC
ACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGT
GGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATG
CTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA
SEQ ID NO: 31
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAAGAAGGATCCTTAGTCTCTATTGAATCTGCTGAGATTA
CACTTTGATGGATGATGCTCTGTTTTTGTTTTCTTGTTCTGTTTTTTCCT
CTGTTGAAATCAGCTTTGTTGCTTGATTTCATTGAAGTTGTTATTCAAGA
ATAAATCAGTTACAATTATGGAATTCGTGGTAACTTTTACTCATCTCCTC
CAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGT
GTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTT
TGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGT
GAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGT
TTTTGTTCATCTGTAGCTTGATATCCTTCTTTGATGCTGATCCATAATTG
TAACTGATTTATTCTTGAATAACAACTTCAATGAAATCAAGCAACAAAGC
TGATTTCAACAGAGGAAAAAACAGAACAAGAAAACAAAAACAGAGCATCA
TCCATCAAAGTGTAATCTCAGCAGATTCAATAGAGACTAAGCTTTTGATT
TTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAA
TTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAA
CCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTT
CTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCT
ACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTA
TGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGA
GATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAA
TAGTCTAGA
SEQ ID NO: 32
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTC
TGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGT
ATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTT
GCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCC
TCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCAT
CTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACATCACGGATCCTT
AGTCTCTATTGAATCTGCTGAGATTACACTTTGATGGATGATGCTCTGTT
TTTGTTTTCTTGTTCTGTTTTTTCCTCTGTTGAAATCAGCTTTGTTGCTT
GATTTCATTGAAGTTGTTATTCAAGAATAAATCAGTTACAATTATGGAAT
TCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTC
GAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTC
GGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTT
GATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAG
CCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAA
TTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTT
CCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTT
GATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAG
CCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTG
AGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCT
AATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTG
ATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTCTTTGATGCTGATC
CATAATTGTAACTGATTTATTCTTGAATAACAACTTCAATGAAATCAAGC
AACAAAGCTGATTTCAACAGAGGAAAAAACAGAACAAGAAAACAAAAACA
GAGCATCATCCATCAAAGTGTAATCTCAGCAGATTCAATAGAGACTAAGC
TTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCG
AACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTA
TGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGC
TTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCT
AGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGT
ATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAG
GAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAG
TAAAAAAATAGTCTAGA
SEQ ID NO: 33
GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATT
CTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTAT
AATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTA
GGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGG
CCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGG
GCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTT
GCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG
AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCAC
TCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTC
TGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGT
ATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTT
GCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCC
TCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCAT
CTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACATCACGGATCCTT
AGTCTCTATTGAATCTGCTGAGATTACACTTTGATGGATGATGCTCTGTT
TTTGTTTTCTTGTTCTGTTTTTTCCTCTGTTGAAATCAGCTTTGTTGCTT
GATTTCATTGAAGTTGTTATTCAAGAATAAATCAGTTACAATTATGGAAT
TCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATG
TTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTC
ATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTG
ACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTC
TTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATAT
CCTTCTTTGATGCTGATCCATAATTGTAACTGATTTATTCTTGAATAACA
ACTTCAATGAAATCAAGCAACAAAGCTGATTTCAACAGAGGAAAAAACAG
AACAAGAAAACAAAAACAGAGCATCATCCATCAAAGTGTAATCTCAGCAG
ATTCAATAGAGACTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATC
AGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGG
ATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTC
AAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCT
TAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTA
GCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATC
GATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATT
GGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA
SEQ ID NO: 34
GTCCATGATGTCTTCAGGGTGGTAGCATTGACTGATGGCATCATAGTTTT
TTTTTTAAAAGTATTTCCTCTATGCATATTATTAGTATCCAATAAATTTA
CTGGTTGTTGTACATAGAAAAAGTGCATTTGCATGTATGTGTTTCTCTGA
AATTTTCCCCAGTTTTTGGTGCTTTGCCTTTGGAGCCAAGTCTCTATATG
TATAAGAAAACTAAGAACAATCACATATATCAAATATTAG
SEQ ID NO: 35
ACGAACTTGTGATCGCGTTGAAAGATTTGAACGCTACATAGAGCTTCTTG
ACGTATCTGGCAATATTGCATCAGTCTTGGCGGAATTTCATGTGACAACA
AGGTTTGCAATTCTTTCCACTATTAGTAGTGCAACGATATACGCAGAGAT
GAAGTGCTGAACAAACATATGTAAAATCGATGAATTTATGTCGAATGCTG
GGACGGGCTTCAGCAGGTTTTGCTTAGT
SEQ ID NO: 36
CCGCGGTTTTCTCTCCATCGCGTCAGAGGCCGGTTTTCGTCGGCATCGAA
GAGGGCCACTCGTTTACCGTCATTTGCCAAAGCAGCGCAAAGGCCCATGA
GTGCGGTGGTTTTGCCAGCACCCCCTTTGAAAGAGCAAAACGTCAAAAGT
TGCATATTCTGATCCCGCCTGTCCTGTGAAACGGAGTGCATTTGTATTTT
TGTTCGTATAAATGTTTTTGTGATTATCGATGAGTAAAAGCGTTGTTACA
CTATTTTTTATTTCAAATTCGTTATAATTAAATTGCAATTGTAGCAATTA
TATTCGGTTTTTCCTGTAAATATACTGTTGATTTCATATCGAGTAGGGCT
AGACTTTAATCTGTCTACCCGGGCACATTTCGTGCTGGAGTATTCAGACC
TTCCGCTTTTTTTGGAGGAAGCTATGTCAAAACACACCAGAGTCACGTCG
AGTGAGACTGCCATCAACCAGCATCGATCCCTGAACGTTGAAGGGTTTAA
GGTCGTGAGTGCCCGTCTGCGATCGGCCGAGTATGAAACCTTTTCCTATC
AAGCGCGCCTGCTGGGACTTTCGGATAGTATGGCAATTCGCGTTGCGGTG
CGTCGCATCGGGGGCTTTCTCGAAATAGATGCACACACCCGAGAAAAGAT
GGAAGCCATACTTCAGTCCATCGGAATACTCTCAAGTAATGTATCCATGC
TTCTATCTGCCTACGCCGAAGACCCTCGATCGGATCTGGAGGCTGTGCGA
GATGAACGTATTGCTTTTGGTGAGGCTTTCGCCGCCCTCGATGGCCTACT
CCGCTCCATTTTGTCCGTATCCCGGCGACGGATCGACGGTTGCTCGCTAT
TGAAAGGTGCCTTGTAGCACTTGACCACGCACCTGACGGGAGAAAATTGG
ATGCCCGATCGCGCTCAAGTAATCATTCGCATTGTGCCAGGAGGTGGAAC
CAAGACCCTTCAGCAGATAATCAATCAGTTGGAGTACCTGTCCCGTAAGG
GAAAGCTGGAACTGCAGCGTTCAGCCCGGCATCTCGATATTCCCGTTCCG
CCGGATCAAATCCGTGAGCTTGCCCAAAGCTGGGTTACGGAGGCCGGGAT
TTATGACGAAAGTCAGTCAGACGATGATAGGCAACAAGACTTAACAACAC
ACATTATTGTAAGCTTCCCCGCAGGTACCGACCAAACCGCAGCTTATGAA
GCCAGCCGGGAATGGGCAGCCGAGATGTTTGGGTCAGGATACGGGGGTGG
CCGCTATAACTATCTGACAGCCTACCACGTCGACCGCGATCATCCACATT
TACATGTCGTGGTCAATCGTCGGGAACTTCTGGGGCACGGGTGGCTGAAA
ATATCCAGGCGCCATCCCCAGCTGAATTATGACGGCTTACGGAAAAAGAT
GGCAGAGATTTCACTTCGTCACGGCATAGTCCTGGATGCGACTTCGCGAG
CAGAAAGGGGAATAGCAGAGCGACCAATCACATATGCTGAACATCGCCGC
CTTGAGCGGATGCAGGCTCAAAAGATTCAATTCGAAGATACAGATTTTGA
TGAGACCTCGCCTGAGGAAGATCGTCGGGACCTCAGTCAATCGTTCGATC
CATTTCGATCGGACCCATCTACCGGCGAACCGGACCGTGCAACCCGACAT
GACAAACAACCGCTTGAACAGCACGCCCGTTTCCAGGAGTCCGCCGGCTC
CAGCATCAAAGCCGACGCACGGATCCGCGTATCATTGGAGAGCGAGCGGA
GTGCCCAACCATCCGCGTCCAAAATCCCTGTAATTGGGCATTTCGGGATT
GAGACTTCCTATGTCGCTGAAGCCAGCGTGCGCAAACGAAGCGGCATTTT
CGGTACTTCTCGCCCGGTGACTGACGTTGCCATGCACACAGTCAAGCGCC
AGCAGCGATCAAAACGACGTAATGACGAGGAGGCAGGTCCGAGCGGAGCA
AACCGTAAAGGATTGAAGGCTGCGCAAGTTGATTCCGAGGCAAATGTCGG
TGAGCAAGACACTCGCGATGACAGCAACAAGGCGGCTGATCCGGTGTCTG
CTTCCATCGGTACCGAGCAACCGGAAGCTTCTCCAAAGCGTCCGCGTGAC
CGTCACGATGGAGAATTGGGTGGACGCAAACGTGCAAGAGGTAATCGTCG
CTCGAGCTCGAGCGGGGGGACCTAGAGACAGGAAGGACCGAATAATGGCC
GCGG
SEQ ID NO: 37
ATGGCTTCTGTGCTGGCTTCTCTGTTTCCAAAACTGGGCTCTTTGGGTAC
TTCAGATCATGCTTCTGTTGTATCCATCAACCTCTTTGTGGCACTCCTTT
GTGCTTGCATCATCATTGGTCATCTCTTGGAGGAGAACCGCTGGGTTAAT
GAGTCCATTACTGCCCTCATAATTGGTTTGTGTACAGGAGTGGTTATCTT
GCTCGTAAGTGGTGGAAAGAGCTCACACCTTCTGGTTTTCAGTGAAGATC
TCTTTTTCATATATGTACTTCCTCCAATCATATTTAATGCAGGGTTTCAG
GTAAAAAAGAAGCAATTTTTCGTAAACTTCATTACTATAATGATGTTCGG
AGCCATTGGTACCCTGGTCTCATGTGCCATTATATCATTAGGTGCCATTC
AAACTTTCAAGAAGTTGGACATTGAATTTCTAGATATTGGGGATTATCTT
GCAATTGGAGCAATATTTGCTGCCACAGATTCCGTCTGCACATTGCAGGT
CCTACATCAGGATGAGACACCCCTCCTTTACAGTCTTGTATTTGGAGAAG
GAGTTGTAAATGATGCTACATCGGTGGTGCTTTTCAATGCTATTCAAAAC
TTCGACCTTACGAGCATGAATCCCAGTATAGCCCTCAGTTTCCTTGGCAA
CTTCTTCTATCTGTTCCTTGCTAGCACTTTACTGGGAGCAGGAACTGGTC
TTCTTAGTGCTTACATTATCAAGAAGCTATATTTTGGCAGGCACTCCACA
GATCGTGAGGTTGCCCTTATGATGCTCATGGCTTACTTATCATACTTGCT
GGCCGAATTATTCTATTTGAGTGGGATTCTCACCGTCTTTTTCTGTGGTA
TTGTAATGTCTCACTACACTTGGCACAATGTGACCGAGAGTTCAAGAGTC
ACTACAAGGCACACTTTTGCAACTTTGTCATTTCTTGCAGAGACTTTCCT
CTTCCTCTATGTCGGCATGGATGCTTTGGATATCGAGAAGTGGAAATTTG
TTGGTGACAGGCCTGGATTATCAATTTCCGTGAGTTCAATACTGATGGGA
CTAATCTTGCTTGGGAGAGCTGCCTTTGTTTTTCCATTATCATTCTTATC
CAACTTAATGAAGAAATCCTCGGAGCAAAAAATTACCTTTAGGCAGCAAG
TGATAATATGGTGGGCAGGTTTGATGAGAGGCGCAGTGTCCATGGCACTG
GCATATAATAAGTTCACTCGTGGGGGACACACTCAACTGCAGGACAATGC
AATAATGATTACCAGCACGATAACCATTGTTCTATTCAGCACAATGGTAT
TCGGTTTAATGACAAAACCCCTTATAAGTCTCCTGCTGCCACCACAGAGG
CAATTGAGTACAGTGTCATCAGGCGCAAATACTCCAAAGTCTCTAACAGC
CCCACTCCTAGGCAGTCGAGAGGACTCTGAAGTTGATTTAAATGTTCCAG
ATCTTCCTCACCCACCAAGTTTGAGGATGCTACTTACCGCACCAAGTCAT
AAAGTGCATCGGTACTGGCGCAAGTTTGACGATGCATTCATGCGCCCTAT
GTTTGGTGGTCGGGGATTTGCTCCTCCTGCCCCTGGTTCTCCAACGGAAC
AGGGTCCATGAGGTACCAATC
SEQ ID NO: 38
ATGGCTTCTGTGCTGGCTTCTCTGTTTCCAAAACTGGGCTCTTTGGGTAC
TTCAGATCATGCTTCTGTTGTATCCATCAACCTCTTTGTGGCACTCCTTT
GTGCTTGCATCATCATTGGTCATCTCTTGGAGGAGAACCGCTGGGTTAAT
GAGTCCATTACTGCCCTCATAATTGGTTTGTGTACAGGAGTGGTTATCTT
GCTCGTAAGTGGTGGAAAGAACTCACACCTTCTGGTTTTCAGTGAAGATC
TCTTTTTCATATATGTACTTCCTCCAATCATATTTAATGCAGGGTTTCAG
GTAAAAAAGAAGCAATTTTTCGTGAACTTCATTACTATAATGATGTTCGG
AGCCATTGGTACCCTGGTCTCATGTGCCATTATATCATTAGGTGCAATTC
AAACTTTCAAGAAGTTGGACATTGAATTTCTAGATATTGGGGATTATCTT
GCAATTGGAGCAATATTTGCTGCCACAGATTCCGTCTGCACATTGCAGGT
CCTACATCAGGATGAGACACCCCTCCTTTACAGTCTTGTATTTGGAGAAG
GAGTTGTAAATGATGCTACATCGGTGGTGCTTTTCAATGCTATTCAAAAC
TTTGACCTTACGAGCGTGAATCCCAGTATAGCCCTCAGTTTCCTTGGCAA
CTTCTTCTATCTGTTCCTTGCTAGCACTTTACTGGGAGCAGGAACTGGTC
TTCTTAGTGCTTACATTATCAAGAAGCTGTATTTTGGCAGGCACTCCACA
GATCGTGAGGTTGCCCTTATGATGCTCATGGCTTACTTATCATACATGCT
GGCTGAACTATTCTATTTGAGTGGGATTCTCACTGTATTTTTCTGTGGTA
TTGTAATGTCTCATTACACTTGGCACAATGTGACCGAGAGTTCAAGAGTC
ACTACAAGGCACGCTTTTGCAACTTTGTCATTTCTTGCAGAGACTTTCCT
CTTCCTCTATGTCGGCATGGATGCTTTGGATATCGAGAAGTGGAAATTTG
TTGGTGACAGGCCTGGATTATCAATTTCCGTGAGTTCAATACTGATGGGA
TTAATCTTGCTGGGGAGAGCTGCCTTTGTTTTTCCATTATCATTCTTCTC
CAACTTAATGAAGAAATCCTCGGAGCAAAAAATTACCTTTAGGCAGCAAG
TGATAATATGGTGGGCAGGTTTGATGAGAGGCGCAGTGTCCATGGCACTG
GCATATAATAAGTTCACTCGTGGGGGACACACTCAACTGCAGGACAATGC
AATAATGATTACCAGCACGATAACCATTGTTCTATTCAGCACAATGGTAT
TCGGTTTAATGACAAAACCCCTTATAAGTCTCCTGCTGCCACCACAGAGG
CAATTGAGTACAGTGTCATCAGGTGCAAATACTCCAAAGTCTCTAACAGC
CCCACTCCTAGGCAGTCGAGAGGACTCTGAAGTTGATTTAAATGTTCCAG
ATCTTCCTCACCCACCAAGTTTGAGGATGCTACTTACCGCACCAAGTCAT
AAAGTGCATCGGTACTGGCGCAAGTTTGACGATGCATTCATGCGCCCTAT
GTTTGGTGGTCGGGGATTTGCTCCTCCTGCCCCTGGTTCTCCAACGGAAC
AGGGTCCATGAGGTACAATC
SEQ ID NO: 39
ATGGAAAATTCGGTACCCAGGACTGTAGAAGAAGTATTCAACGATTTCAA
AGGTCGTAGAGCTGGTTTAATCAAAGCACTAACTACAGATGTCGAGAAGT
TTTATCAATCGTGTGATCCTGAAAAGGAGAACTTGTGTCTCTATGGGCTT
CCTAATGAAACATGGGAAGTAAACCTCCCTGTAGAGGAGGTGCCTCCAGA
ACTTCCGGAGCCAGCATTGGGCATAAACTTCGCACGTGATGGAATGCAAG
AGAAAGACTGGTTATCACTTGTTGCTGTTCACAGTGATTCATGGCTGCTT
TCTGTTGCATTTTACTTTGGTGCAAGGTTTGGGTTCGGCAAGAGTGAAAG
GAAGAGGCTTTTCCAAATGATCTCCCAACAGTGTTTGAAGTTGTTACCGG
AGCTGCTAAACAGACACGTGATCCCCCTCACAACAATAGCAACAAAAGCA
AATCAAGTGGAAAGCCTCGACAGCCAGAGTCCCAACTCAAGGCAGTAAAG
GTGTCTCCACCTAAAATGGAGAACGACAGTGGGGAGGAGGAAGAAGAAGA
AGAGGATGAACAAGGAGCAACTCTCTGTGGAGCTTGTGGTGATAATTATG
CCACTGATGAATTCTGGATTTGCTGTGATATTTGTGAGAGATGGTTCCAT
GGCAAATGTGTGAAGATTACCCCAGCAAAAGCTGAGCATATCAAGCAGTA
CAAGTGTCCTAGTTGCAGTAGCAAGAGAGCTAGAGTTTAA
SEQ ID NO: 40
TGACATCTGCCAATAAAGCCAAGAATAATTGGCATTAACATGACCAAAAA
AATGGTTTGGCAGCATTAAGTCAAATAAAAAAGCTACTTTAATATAAAAT
AATATTAAAATGCTTAATAACCAACAGTTTATAAGAAGGTTAATGTTAAC
ATGGATGAGGAATGACCAAAAGGGGAATTATATATTAACCTTTAAATCAA
TCTAATTCTCTCTTTTTGTTTCTAGCTATATTTACTCGATAGATAAACTC
TCTTACTTGACGAATTTTTTGATACAAGAAGACATATTTCATCATGATTT
TAATTCGTCGTGTCAAATTTATTAAATAGTTTAATTTTAATCGTAAATTT
AGATATGAAATTTAAAAAAAAATAAATATATACATATTTGAAGAATACAT
AAAAAGTACATATAAATCACAAATATTTAATAATTCAAGATATTAAAACA
CATAGAAAAATAATTACTTACAAAGAAATTCTTATTTGAATCCTCTAAAT
TCGAGAAGTGCAACACAAACTGAGACGAAGAAAATGAATAATATTTGATA
AGAAATTTATTATAATTGAATGACCATTTAAGTAATTACGGGTAATAACA
ACACAATAAGGAACTGTAGTCATTTTTAATACATGGCAAGGAATATGAGA
GTGTGATGAGTCTATAAATAGAAGGCTTCATTAGTGTAGAGGAGTCACAA
ACAAGCAATACACAAATAAAATTAGTAGCTTAAACAAGATG
SEQ ID NO: 56
TTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTC
TACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTA
GATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTTTATTA
TTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCT
TAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGT
CAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTC
SEQ ID NO: 94
TGGCAGGATATATGAGTGTGTAAAC
SEQ ID NO: 95
TTGGCAGGATATATCCCTCTGTAAAC
Rommens, Caius, Yan, Hua, Richael, Craig, Ye, Jingsong, Menendez-Humara, Jaime, Brinkerhoff, W. Leigh, Swords, Kathy M. M.
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