Patent application title: Pesticidal Nucleic Acids and Proteins and Uses Thereof
Inventors:
IPC8 Class: AC07K14325FI
USPC Class:
800301
Class name: Plant, seedling, plant seed, or plant part, per se higher plant, seedling, plant seed, or plant part (i.e., angiosperms or gymnosperms) pathogen resistant plant which is transgenic or mutant
Publication date: 2016-07-14
Patent application number: 20160200778
Abstract:
The invention provides compositions comprising polynucleotide molecules
encoding certain pesticidal polypeptides which exhibit plant parasitic
nematode and/or insect control properties, and are particularly directed
to controlling plant parasitic pest species of nematodes and insects
known to infest crop plant species. Methods for controlling pests are
disclosed in which the toxic proteins are provided in the diet of the
targeted plant pests. The invention also provides compositions such as
nucleic acids, proteins, and plant and bacterial cells, plants, and seeds
containing the nucleic acid and protein compositions, as well as methods
and kits for identifying, detecting, and isolating the compositions of
the present invention. The invention further provides a method of
producing crops from recombinant seeds which contain the polynucleotide
molecules encoding the pesticidal polypeptides of the present invention.Claims:
1-9. (canceled)
10. A DNA construct comprising a polynucleotide operably linked to a heterologous promoter, wherein said polynucleotide encodes a pesticidal polypeptide that comprises an amino acid sequence at least 95% identical to SEQ ID NO:42.
11. The DNA construct of claim 10, wherein said polynucleotide encodes a pesticidal polypeptide that comprises an amino acid sequence at least 99% identical to SEQ ID NO:42.
12. The DNA construct of claim 10, wherein said polynucleotide encodes a pesticidal polypeptide that comprises the amino acid sequence as set forth in SEQ ID NO:42.
13. The DNA construct of claim 10, wherein said polynucleotide is codon-optimized for expression in a plant.
14. The DNA construct of claim 13, wherein said polynucleotide comprises the nucleic acid sequence as set forth in SEQ ID NO:43.
15. A host cell comprising a DNA construct that comprises a polynucleotide operably linked to a heterologous promoter, wherein said polynucleotide encodes a pesticidal polypeptide that comprises an amino acid sequence at least 95% identical to SEQ NO:42.
16. The host cell of claim 15, wherein said polynucleotide encodes a pesticidal polypeptide that comprises an amino acid sequence at least 99% identical to SEQ ID NO:42.
17. The host cell of claim 15, wherein said polynucleotide encodes a pesticidal polypeptide that comprises the amino acid sequence as set forth in SEQ ID NO:42.
18. The host cell of claim 15, wherein said host cell is a bacterial cell or a plant cell.
19. The host cell of claim 18, wherein said bacterial cell is selected from the group consisting of an Agrobacterium, a Bacillus, an Escherichia, a Salmonella, a Pseudomonas, and a Rhizohium cell, and wherein said plant cell is selected from the group consisting of a alfalfa, banana, barley, bean, broccoli, cabbage, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and a wheat plant cell.
20. A plant, or part thereof, comprising a polynucleotide encoding a pesticidal polypeptide that comprises an amino acid sequence at least 95% identical to SEQ ID NO:42.
21. The plant, or part thereof, of claim 20, wherein said polynucleotide comprises the nucleic acid sequence as set forth in SEQ ID NO:43.
22. The plant, or part thereof, of claim 20, wherein said plant is elected from the group consisting of a alfalfa, banana, barley, bean, broccoli, cabbage, canola, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and a wheat plant, and wherein said part is selected from the group consisting of a leaf, a stem a flower, a sepal, a fruit, a root, or a seed.
23. A method of controlling a pest infection of a plant, said method comprising providing in a diet of said pest a plant, or part thereof, said plant or part comprising a polynucleotide encoding a pesticidal polypeptide that comprises an amino acid sequence at least 95% identical to SEQ ID NO:42.
24. The method of claim 23, wherein said pest is an insect or a nematode.
25. The method of claim 24, wherein said insect is an insect from the insect order selected from the group consisting of Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, and Trichoptera, and wherein said nematode is selected from the group consisting of Acontylus, Anguina, Aorolaimus, Aphasmatylenchus, Aphelenchoides, Aphelenchus, Atalodera, Atylenchus, Bakernema Belonolaimus, Brachydorus, Bursaphelenchus, Cacopaurus, Caloosia, Carphadorus, Criconema, Criconemella, Cryphodera, Ditylenchus, Dolichodorus, Eutylenchus, Globodera, Gracilacus, Helicotylenchus, Hemicriconemoides, Hemicycliophora, Heterodera, Hirschmanniella, Histotylenchus, Hoplolaiinus, Hoplotylus, Longidorus, liacrotrophurus, Meloidodera, Meloidogyne, Merlinius, Morulaimus, Nacobbus, Nothanguina, Nothotylenchus, Paralongidorus, Paratrichodorus, Paratrophurus, Paratylenchus, Peltamigratus, Pratylenchoides, Pratylenchus, Psilenchus, Radopholoides, Radopholus, Rhadinaphelenchus, Rototylenchus, Rotylenchoides, Rotylenchus, Sarisodera, Scutellonema, Sphaeronema, Subanguina, Telotylenchoides, Telotylenchus, Trichotylenchus, Trophonerna, Trophotylenculus, Trophurus, Tylenchorhynchus, Tylenchulus, Tylenchus, Tylodorus, Xiphinema, and Zygotylenchus nematode.
26. The method of claim 23, said method further comprising providing in the diet of said pest a pesticidally effective amount of one or more other toxic agents selected from the group consisting of methylketone synthase, dsRNA, a Cry protein, a VIP protein, and a chemical nematicide.
27. The method of claim 23, said method further comprising providing in a diet of said pest a pesticidally effective amount of one or more pesticidal polypeptides, wherein said one or more pesticidal polypeptides comprise SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ II) NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, or SEQ ID NO:60, or a pesticidal fragment thereof.
28. The method of claim 27, wherein the pesticidally effective amount of said one or more pesticidal polypeptides is provided by the plant, which is a recombinant plant, a part of the plant, or a product of the plant or the plant part.
29. The method of claim 27, wherein the pesticidally effective amount of said one or more pesticidal polypeptides is provided in one or more formulations topically applied on the plant or a part of the plant, said one or more formulations comprising bacterial cells, spores, or parasporal crystals that comprise said one or more pesticidal polypeptides.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Ser. No. 61/441,697 and U.S. Provisional Application Ser. No. 61/441,709, both filed Feb. 11, 2011, which are incorporated herein by reference.
INCORPORATION OF SEQUENCE LISTING
[0002] The Sequence Listing accompanying this application is contained within the computer readable file "38-21(57560) SEQUENCE LISTING_ST25.txt" submitted electronically and contemporaneously with the filing of this application through the USPTO EFS-Web. The file is 105 kilobytes (measured in MS-Windows), was created on 19 Jan. 2012, and is incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention relates to novel polynucleotide and protein compositions that, when expressed and or produced in plants, impart resistance to plant pathogenic nematodes and insect infestation. The polynucleotides and proteins can be expressed in plant and bacterial cells, and the plant cells can be regenerated into transgenic (recombinant) plants, plant tissues, plant parts, and seeds. Compositions derived from such plants, plant materials, and seed that contain detectable amounts of such polynucleotides and proteins are included within the scope of the invention. The invention also relates to compositions and methods for controlling plant pathogenic nematodes and insect pests of crop plants.
BACKGROUND OF THE INVENTION
[0004] The increasing human population will require higher yields of food, feed, and fiber from crop plants on decreasing amounts of arable land. Several types of insects and nematodes are known to reduce yield of crops produced from plants. Plant pests damage plant parts, including roots, developing flower buds, flowers, leaves, stems, and seeds, which leads to lower yields.
[0005] Traditional approaches for controlling plant pests have used chemical control agents and construction of inter-specific hybrids between crops and their wild-type relatives as sources of resistant germplasm. Chemical pest control agents, although effective, have several disadvantages. Many chemical control agents are expensive to manufacture, and are characterized as pollutants because they persist in the environment as a result of their resistance to microbial degradation. Chemical control agents require on-farm formulation, which increases the safety risk to the farmer due to the exposure to chemical agent formulations. The chemical agent formulations have to be applied at least once and often, more than once per growing season, increasing the carbon footprint related to these compositions. Methods and compositions employing plant biotechnology pest control agents are also effective means for controlling plant pests, for instance through plant expression of one or more pest control agents that are generally selectively toxic to a particular target pest when ingested by the pest. Unlike chemical agents, biotech approaches have been demonstrated to be environmentally friendly, have no known safety risks when used by farmers, and are economical in terms of carbon footprint impact and ease of use for deployment by the farmer. However, there are only a few examples of such biotech compositions and methods for controlling such pests, and even fewer if any examples of any biotechnology approaches that have demonstrated efficacy in controlling plant pathogenic nematodes. Thus, there is a need for new compositions and methods for protecting plants from such pest infestation, generally for the purpose of maintaining and enhancing yields of crops produced from such plants, and for sustaining and providing food, feed and fiber for the increasing human population.
SUMMARY OF THE INVENTION
[0006] Polynucleotide molecules are provided encoding exemplary pesticidal polypeptides as set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, and SEQ ID NO:60. Polypeptides having an amino acid sequence exhibiting from at least about 45% to about 99.9% identity to the pesticidal protein (polypeptide) sequences as set forth in any of the foregoing protein sequences (any percentage in between 45 and 99.9) and exhibiting substantially equivalent (biologically functional equivalent) pesticidal activity as any one of these sequences are specifically contemplated. Fragments of these polypeptide sequences that exhibit the requisite pesticidal activity are intended to be within the scope of the present invention. Such polynucleotides may be extracted and/or obtained directly from a host cell or made artificially through various means of synthesis, and in either case, are considered to be recombinant polynucleotides.
[0007] Polynucleotides containing one or more nucleotide sequence segments encoding the pesticidal proteins of the present invention are provided, which may be operably linked to a heterologous promoter that initiates expression of the sequence region in a designated host cell, resulting in the production or manufacture of the pesticidal protein in the host cell. The promoter may include a plant-expressible promoter, a promoter that functions in one or more species of bacteria, and a yeast functional promoter, or combinations thereof. The plant-expressible promoter may include any number of promoters known in the art, including but not limited to corn sucrose synthetase 1 promoter, corn alcohol dehydrogenase 1 promoter, corn light harvesting complex promoter, corn heat shock protein promoter, pea small subunit RuBP carboxylase promoter, Ti plasmid mannopine synthase promoter, Ti plasmid nopaline synthase promoter, petunia chalcone isomerase promoter, bean glycine rich protein 1 promoter, Potato patatin promoter, lectin promoter, CaMV 35S promoter, FMV promoter, ubiquitin promoters promoter, and the S E9 small subunit RuBP carboxylase promoter.
[0008] Isolated polynucleotide segments are provided for use as probes and/or primers, which may be from about 20 to about 1000 contiguous nucleotides in length or any length in between twenty and one thousand contiguous nucleotides, and exhibit at least about 90% identity to the same contiguous length of nucleotides as set forth in any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63, or the complement of any of the foregoing polynucleotide sequences.
[0009] In another aspect of the invention, polynucleotides encoding any of the pesticidal polypeptides set forth above are provided in recombinant expression cassettes. The expression cassettes can be provided in vectors for use in replicating, maintaining and transferring the nucleic acid component encoding the pesticidal proteins of the present invention. The vectors of the present invention contain at least a sequence region that encodes the polypeptide as set forth above. The vector includes a plasmid, baculovirus, artificial chromosome, virion, cosmid, phagemid, phage, or viral vector.
[0010] Host cells may be any appropriate transgenic host cell including but not limited to microbial cells (microorganisms) such as an Agrobacterium, a Bacillus, an Escherichia, a Salmonella, a Pseudomonas, a Rhizobium bacterial cell, a yeast cell such as a pichia yeast or saccharomyces species yeast cell, or a plant cell. Vectors as described above can be provided in a transgenic microbial host cell. The transgenic microbial host cell includes a prokaryotic or eukaryotic host cell. The transgenic prokaryotic host cell is a bacterial cell and the transgenic eukaryotic host cell is a plant or a fungal/yeast cell. The transgenic bacterial cell includes a recombinant bacterium including a Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Bacillus laterosperous, Escherichia, Salmonella, Agrobacterium, Rhizobium, or Pseudomonas cell. The transgenic plant host cell includes a monocotyledonous or dicotyledonous plant cell and may include any plant cell from the Group of Plants or Plant Group set forth below. To the extent that a microbial cell is a plant cell, the cell can be obtained from any plant, plant tissue, plant part or seed from a plant selected from the group consisting of any of the following, including but not limited to barley, bean, broccoli, cabbage, canola (rapeseed), carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, coffee, corn (including sweet corn), clover, cotton, a cucurbit, cucumber, deciduous trees (including but not limited to banana, citrus, eucalyptus, nut trees (including but not limited to hickory, pecan, and walnut trees), oak trees (including but not limited to live oak, pin oak, and post oak trees), olive, palm (including coconut palm), poplar, sweet gum, and rootstocks of all of the preceding trees), eggplant, evergreen trees (including but not limited to Douglas fir), flax, garlic, grape, grasses (including but not limited to alfalfa, pasture grass, switchgrass, and turf grass), hops, leek, lettuce, millets, melons (including but not limited to cantaloupe, honeydew melon, and watermelon), oat, onion, pea, peanut, pepper, pigeonpea, pine (including Loblolly pine, Radiata pine, and Southern pine), potato, pumpkin, radish, rice, rye, safflower, shrub, sorghum, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet potato, tea, tobacco, tomato, triticale, or wheat. The aforementioned are referenced herein as the "Group of Plants" or the "Plant Group".
[0011] Recombinant plants, plant tissue, plant parts, or seed contain the polynucleotides of the present invention and express the proteins of the present invention from such polynucleotides. The plant part is a leaf, a stem a flower, a sepal, a fruit, a root, or a seed. Products produced from a recombinant plant of the present invention are also contemplated, and can include at least any of the following: oil, meal, lint and seed of the recombinant plant. The polynucleotides and proteins of the present invention are present in a detectable amount in the plants and plant products, and are useful at least as markers for tracking the presence of seeds and plant tissues containing the polynucleotide and proteins through trade and commerce, in fields of crops, and in various embodiments referenced herein.
[0012] There is provided a method of detecting and/or isolating in or from a biological sample, a polynucleotide molecule encoding a pesticidal polypeptide of the present invention in which the steps of the method include (i) selecting a pair of oligonucleotide primers that produce an amplicon encoding all or a representative amount of the pesticidal polypeptide of the present invention when used together in an amplification reaction with the biological sample containing the polynucleotide; (ii) producing the amplicon from the polynucleotide; (iii) detecting and/or isolating the amplicon; and (iv) generating nucleotide sequence information corresponding to the amplicon to identify and confirm the presence (or absence) of a segment of a polynucleotide molecule encoding all or a representative amount of the pesticidal polypeptide. Alternatively, the detecting and/or isolating step can be conducted by providing a polynucleotide probe derived from a sufficient length of DNA or RNA encoding the pesticidal polypeptide that hybridizes under specific or under stringent hybridization conditions to such a polynucleotide encoding a pesticidal polypeptide of the present invention.
[0013] Methods of controlling or killing a target lepidopteran pest, coleopteran pest, or plant pathogenic nematode pest population are provided and include contacting the pest population with a pesticidally-effective amount of the polypeptide as set forth above. The "lepidopteran pest population" includes Spodoptera frugiperda, Spodoptera exigua), Mamestra configurata, Agrotis ipsilon, Trichoplusia ni, Pseudoplusia includens, Anticarsia gemmatalis, Hypena scabra, Heliothis virescens, Agrotis subterranea, Pseudaletia unipuncta, Agrotis orthogonia, Ostrinia nubilalis, Amyelois transitella Crambus caliginosellus, Herpetogramma licarsisalis, Homoeosoma electellum, Elasmopalpus lignosellu, Cydia pomonella, Endopiza viteana, Grapholita molesta, Suleima helianthana, Plutella xylostella, Pectinophora gossypiella, Lymantria dispar, Blatta orientalis, Blatella asahinai, Blattella germanica, Supella longipalpa, Periplaneta americana, Periplaneta brunnea, Leucophaea maderae, Alabama argillacea, Archips argyrospila, A. rosana, Chilo suppressalis, Cnaphalocrocis medinalis, Crambus caliginosellus, C. teterrellus, Diatraea grandiosella, D. saccharalis, Earias insulana, E. vittella, Helicoverpa armigera, H. zea, Heliothis virescens, Herpetogramma licarsisalis, Lobesia botrana, Pectinophora gossypiella, Phyllocnistis citrella, Pieris brassicae, P. rapae, Plutella xylostella, Spodoptera exigua, S. litura, S. frugiperda, Tuta absoluta. The "coleopteran pest population" includes Anthonomus grandis, Lissorhoptrus oryzophilu, Sitophilus granaries, Sitophilus oryzae, Hypera punctata, Sphenophorus maidis, Leptinotarsa decemlineata, Diabrotica virgifera virgifera, Diabrotica barberi, Diabrotica undecimpunctata howardi, Chaetocnema pulicaria, Phyllotreta cruciferae, Colaspis brunnea, Oulema melanopus, Zygogramma exclamationis, Epilachna varivestis, Popillia japonica, Cyclocephala boreali, Cyclocephala immaculata, Rhizotrogus majalis, Phyllophaga crinita, Ligyrus gibbosus, Melanotus spp., Conoderus spp., Limonius spp., Agriotes spp., Ctenicera spp., Aeolus spp., Eleodes spp. The "plant pathogenic nematode population" includes Heterodera glycines (soybean cyst nematode), Heterodera schachtii (beet cyst nematode), Heterodera avenae, Globodera rostochiensis, Globodera pailida, Pratylenchus zeae (a root knot nematode), Meloidogyne javanica, Pratylenchus brachyurus (a root knot nematode), Meloidogyne hapla, Meloidogyne incognita.
[0014] An alternative method for controlling such plant pest infection includes providing a pest inhibitory amount of a pesticidal polypeptide of the present invention to a pest susceptible to the polypeptide, thereby controlling the pest. The pest is an insect or a nematode. The insect may be any insect within the taxonomical orders including Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, or Trichoptera (hereinafter, the "Insect Orders"). The nematode may be from any genus of nematodes referred to as Acontylus, Anguina, Aorolaimus, Aphasmatylenchus, Aphelenchoides, Aphelenchus, Atalodera, Atylenchus, Bakemema, Belonolaimus, Brachydorus, Bursaphelenchus, Cacopaurus, Caloosia, Carphodorus, Criconema, Criconemella, Cryphodera, Ditylenchus, Dolichodorus, Eutylenchus, Globodera, Gracilacus, Helicotylenchus, Hemicriconemoides, Hemicycliophora, Heterodera, Hirschmanniella, Histotylenchus, Hoplolaimus, Hoplotylus, Longidorus, Macrotrophurus, Meloidodera, Meloidogyne, Merlinius, Morulaimus, Nacobbus, Nothanguina, Nothotylenchus, Paralongidorus, Paratrichodorus, Paratrophurus, Paratylenchus, Peltamigratus, Pratylenchoides, Pratylenchus, Psilenchus, Radopholoides, Radopholus, Rhadinaphelenchus, Rototylenchus, Rotylenchoides, Rotylenchus, Sarisodera, Scutellonema, Sphaeronema, Subanguina, Telotylenchoides, Telotylenchus, Trichotylenchus, Trophonema, Trophotylenculus, Trophurus, Tylenchorhynchus, Tylenchulus, Tylenchus, Tylodorus, Xiphinema, or Zygotylenchus (hereinafter, the "Nematode Species"). In related embodiments, the nematode species includes cyst and related nematodes such as Heterodera glycines (soybean cyst nematode), Heterodera schachtii (beet cyst nematode), Heterodera avenae (cereal cyst nematode), and Globodera rostochiensis and Globodera pailida (potato cyst nematodes), Pratylenchus zeae, Meloidogyne javanica, Pratylenchus brachyurus, Meloidogyne hapla, or Meloidogyne incognita (hereinafter, the "Cyst Nematode" group). The pest inhibitory amount of the pesticidal polypeptide is provided in the diet of the pest, and the diet of the pest can be a part of a recombinant plant, seed of such plant, or product of the plant. The pest inhibitory amount of the polypeptide may also be provided in a topical formulation to a plant. Such formulation could include a preparation containing bacterial cells, bacterial spores, and parasporal crystals which contain or are producing one or more of the polypeptides/toxic agents of the present invention in a sufficient amount to inhibit the pest infestation of the plant to which the formulation is applied. A formulation for controlling nematode or insect species within the scope of the present invention may consist of recombinant bacterial cells and/or sporeswhich may be producing the toxic proteins of the present invention, or parasporal crystals that contain pesticidal amounts of the polypeptide. The bacterial cells, spores, or parasporal crystals are typically from Bacillus species. Antibodies are contemplated that specifically bind to a polypeptide having the amino acid sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, and/or SEQ ID NO:60, or a peptide, or an epitope derived therefrom. Particularly, purified antibodies that specifically bind to one or more of the polypeptides of the present invention, or to a peptide or epitope derived from the proteins of the present invention are contemplated.
[0015] Such antibodies are useful at least in methods of detecting pesticidal polypeptides such as those set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, and/or SEQ ID NO:60 in a biological sample. A method of detecting such proteins could include the steps of contacting the biological sample with an antibody that specifically binds to one or more of the proteins of the present invention, and detecting the binding of the antibody to the pesticidal polypeptide. Alternatively, proteins of the present invention, or proteins that are substantially related to the proteins of the present invention can be detected in or isolated from a biological sample either by directly identifying the protein in the sample using for example, antibodies as indicated above, or by screening for the presence of a polynucleotide encoding the pesticidal protein. Detecting the polynucleotide encoding such protein could include the steps of: i) selecting a pair of primers that produce an amplicon encoding the pesticidal protein when used together in an amplification reaction with the polynucleotide; ii) producing the amplicon by using the polynucleotide as a template in the amplification reaction; iii) detecting/isolating the the amplicon; iv) generating DNA sequence information corresponding to the amplicon to confirm that the amplicon encodes the pesticidal protein; and v) testing the pesticidal protein to confirm pesticidal activity. Alternatively, a method for detecting the protein of the present invention, or a related pesticidal protein such as a .delta.-endotoxin polypeptide, in a biological sample could include the steps of: i) obtaining a biological sample suspected of containing a .delta.-endotoxin polypeptide; ii) contacting the sample with an antibody that specifically binds to the polypeptide under conditions effective to allow the formation of immune complexes; and iii) detecting the immune complexes so formed. Another alternative method for detecting a target pesticidal polypeptide of the present invention in a sample may include the steps of: i) contacting the sample with an antibody that specifically binds the target pesticidal polypeptide; ii) detecting the binding of the antibody to the target in the sample; and iii) identifying the target as a pesticidal polypeptide exhibiting at least 90% amino acid sequence identity to any one of the proteins set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, and/or SEQ ID NO:60.
[0016] Detection methods can be conducted using reagents and instructions packaged together in kit form and are useful for detecting the proteins and polynucleotides of the present invention. Such kits could include a first reagent or antibody that binds specifically to the polypeptide, or specifically to a peptide or an epitope derived therefrom; and a second reagent such as a control polypeptide corresponding to any of the proteins as set forth in any of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, and/or SEQ ID NO:60, or a peptide, or an epitope derived therefrom.
[0017] In another aspect of the present invention, there is provided a method of preparing insect resistant plants. Such plants can be prepared by contacting a recipient plant cell with a transgene that encodes one or more of the polypeptides of the present invention under conditions permitting the uptake of the transgene by the cell, and selecting a recipient cell in which the transgene has been incorporated into the cell genome, and regenerating a plant from the selected recipient cell. The regenerated plant is confirmed to be a fertile transgenic plant exhibiting pest resistance, and the pest resistance includes resistance to plant pathogenic nematode infestation and one other pest resistance selected from resistance against to a coleopteran insect or to a lepidopteran insect. The contacting step includes any one or ore of the methods known in the art, including microprojectile bombardment, electroporation or Agrobacterium-mediated plant cell transformation. The regenerated plant is resistant to at least one of the members of the plant parasitic nematode group including Heterodera species, Globodera species, Meloidogyne species, Rotylenchulus species, Hoplolaimus species, Belonolaimus species, Pratylenchus species, Longidorus species, Paratrichodorus species, Ditylenchus species, Xiphinema species, Dolichodorus species, Helicotylenchus species, Radopholus species, Hirschmanniella species, Tylenchorhynchus species, or Trichodorus species.
[0018] Transgenic seed containing one or more polynucleotide segments encoding one or more of the proteins of the present invention may be produced comprising the steps of: transforming a plant with a transgene that encodes the polypeptide as set forth above, the transgene operably linked to a promoter that expresses the transgene in a plant, thereby obtaining a fertile transgenic plant comprising the transgene; and growing the plant under appropriate conditions to produce the transgenic seed.
[0019] Progeny of any generation of a pest resistance-enhanced fertile transgenic plant can be produced from such transgenic plants and seeds of the foregoing plants and seed, wherein the progeny contain the polynucleotide and encode the protein(s) of the present invention, and has enhanced pest resistance against a coleopteran insect, lepidopteran insect, or a plant pathogenic nematode relative to the corresponding non-transgenic plant.
[0020] Pest resistant plants can be produced by following the method of: (a) crossing a pest resistant plant comprising a transgene that encodes the polypeptide as set forth above with another plant; (b) obtaining at least one progeny plant derived from the cross of (a); and (c) selecting progeny that comprises the transgene, wherein the progeny is resistant against a coleopteran insect, lepidopteran insect, or a plant pathogenic nematode.
[0021] Seed can be produced from the plants of the present invention. Seed containing a polynucleotide molecule encoding one or more of the proteins of the present invention, whether homogyzous or heterozygous for the particular transgenic allele, can be packaged for planting in a field, and a crop can be produced from the planted seed. The crop from such plants can be harvested, and if seed of the harvested generation are the crop (such as soybean, rice, wheat, canola or corn and the like), at least 50% of the harvested crop are seed containing the polynucleotide molecule.
[0022] Commodity products (or biological samples) containing a plant or plant part as set forth above that can be shown to contain a detectable amount of a polypeptide having the amino acid sequence of any of the proteins of the present invention, or polynucleotides encoding any such protein. The detection of the polypeptide or the polynucleotide in the commodity (or biological sample) is determinative of the presence of the plant or plant part in the commodity (or biological sample), and all such commodity products in which the polypeptide is detectable to a level of at least about (i) one part per million, (ii) or one nanogram per gram fresh weight of tissue, are within the scope of the present invention. A plant cell of the present invention may be regenerated into a recombinant plant which can produce a plant part containing any of the proteins of the present invention. The plant part includes a leaf, a stem a flower, a sepal, a fruit, a root, or a seed. Products produced from a recombinant plant or plant part contain a detectable amount of any one of the proteins of the present invention, or polynucleotide segments encoding such proteins. Such products include oil, meal, lint and seed of such recombinant plants. The detectable amount of the proteins and/or polynucleotides are useful as molecular markers for tracking and/or identifying the presence of seeds and plant tissues of the present invention as these are moved through commerce.
[0023] The proteins of the present invention originate from Bacillus thuringiensis species of bacteria, and as such, are likely to be characterized as delta-endotoxins, and are typically produced from a recombinant polynucleotide. Such delta endotoxin proteins will have an amino acid sequence that exhibits at least from about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% amino acid sequence identity to the amino acid sequence as set forth in any of the sequences shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, and SEQ ID NO:60. Each such protein will preferably include at least 50, or from about 50 to about 100, or from about 50 to about 300 contiguous amino acids present in any full length protein sequence set forth in the sequences referenced above, and the toxin proteins are preferably encoded by a polynucleotide segment that hybridizes under stringent conditions to the polynucleotide coding sequences as set forth in any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63.
[0024] Compositions containing the proteins of the present invention are provided in an agriculturally-acceptable carrier. The composition may contain a recombinant Bacillus thuringiensis cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate, or cell pellet in which at least a pest inhibitory amount of one or more of the proteins of the present invention are provided, and the composition can be provided in the form of a powder, dust, pellet, granule, spray, emulsion, colloid, or solution. The composition may be prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of recombinant Bacillus thuringiensis cells or spore ctystals containing one or more of the proteins of the present invention. The pesticidal composition preferably contains from about 1% to about 99% by weight of one or more of the pesticidal proteins described herein.
[0025] The proteins of the present invention can be obtained in substantially concentrated and/or purified form by a process which may include the steps of i) culturing recombinant Bacillus thuringiensis cells containing one or more recombinant polynucleotide as set forth above under conditions effective to produce the pesticidal protein, and obtaining the pesticidal polypeptide so produced. The polypeptide will preferably contain the contiguous amino acid sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, and SEQ ID NO:60.
BRIEF DESCRIPTION OF THE SEQUENCES
[0026] SEQ ID NO 1 represents a native Bt nucleotide sequence encoding a ET34 protein.
[0027] SEQ ID NO 2 represents an amino acid sequence translation of SEQ ID NO 1.
[0028] SEQ ID NO 3 represents an artificial sequence encoding a ET34 protein.
[0029] SEQ ID NO 4 represents an amino acid sequence translation of SEQ ID NO 3 from nucleotide position 1 through nucleotide position 378.
[0030] SEQ ID NO 5 represents a nucleotide sequence encoding a P139 secretion signal peptide (nucleotide position 1-75) fused in frame to a native Bt nucleotide sequence encoding a ET34 protein (nucleotide position 76-450).
[0031] SEQ ID NO 6 represents the amino acid sequence translation of SEQ ID NO 5.
[0032] SEQ ID NO 7 represents a nucleotide sequence encoding a P139 secretion signal peptide (nucleotide position 1-75) fused in frame to a synthetic nucleotide sequence encoding a ET34 protein (nucleotide position 76-450).
[0033] SEQ ID NO 8 represents the amino acid sequence translation of SEQ ID NO 7.
[0034] SEQ ID NO 9 represents the native Bt nucleotide sequence encoding a TIC1506 protein.
[0035] SEQ ID NO 10 represents the amino acid sequence translation of SEQ ID NO 9.
[0036] SEQ ID NO 11 represents an artificial nucleotide sequence encoding a TIC1506 protein.
[0037] SEQ ID NO 12 represents the amino acid sequence translation of SEQ ID NO 11.
[0038] SEQ ID NO 13 represents the native Bt nucleotide sequence encoding a TIC1501 protein.
[0039] SEQ ID NO 14 represents the amino acid sequence translation of SEQ ID NO 13.
[0040] SEQ ID NO 15 represents an artificial nucleotide sequence encoding a TIC1501 protein.
[0041] SEQ ID NO 16 represents the amino acid sequence translation of SEQ ID NO 15.
[0042] SEQ ID NO 17 represents the native Bt nucleotide sequence encoding a TIC1503 protein.
[0043] SEQ ID NO 18 represents the amino acid sequence translation of SEQ ID NO 17.
[0044] SEQ ID NO 19 represents an artificial nucleotide sequence encoding a TIC1503 protein.
[0045] SEQ ID NO 20 represents the amino acid sequence translation of SEQ ID NO 19.
[0046] SEQ ID NO 21 represents a native Bt nucleotide sequence encoding a TIC614 protein.
[0047] SEQ ID NO 22 represents an amino acid sequence translation of SEQ ID NO 21.
[0048] SEQ ID NO 23 represents an artificial nucleotide sequence encoding a TIC614 protein.
[0049] SEQ ID NO 24 represents an amino acid sequence translation of SEQ ID NO 23.
[0050] SEQ ID NO 25 represents a nucleotide sequence encoding a TIC615 protein.
[0051] SEQ ID NO 26 represents the amino acid sequence translation of SEQ ID NO 25.
[0052] SEQ ID NO 27 represents an artificial nucleotide sequence encoding a TIC615 protein.
[0053] SEQ ID NO 28 represents the amino acid sequence translation of SEQ ID NO 27.
[0054] SEQ ID NO 29 represents the native Bt nucleotide sequence encoding a TIC1277 protein.
[0055] SEQ ID NO 30 represents the amino acid sequence translation of SEQ ID NO 29.
[0056] SEQ ID NO 31 represents an artificial nucleotide sequence encoding a TIC1277 protein.
[0057] SEQ ID NO 32 represents the amino acid sequence translation of SEQ ID NO 31.
[0058] SEQ ID NO 33 represents the native Bt nucleotide sequence encoding a TIC TIC1278 protein.
[0059] SEQ ID NO 34 represents the amino acid sequence translation of SEQ ID NO 33.
[0060] SEQ ID NO 35 represents an artificial nucleotide sequence encoding a TIC TIC1278 protein.
[0061] SEQ ID NO 36 represents the amino acid sequence translation of SEQ ID NO 35.
[0062] SEQ ID NO 37 represents the native Bt nucleotide sequence encoding a TIC TIC1310 protein.
[0063] SEQ ID NO 38 represents the amino acid sequence translation of SEQ ID NO 37.
[0064] SEQ ID NO 39 represents an artificial nucleotide sequence encoding a TIC1310 protein.
[0065] SEQ ID NO 40 represents the amino acid sequence translation of SEQ ID NO 39.
[0066] SEQ ID NO 41 represents the native Bt nucleotide sequence encoding a TIC TIC1311protein.
[0067] SEQ ID NO 42 represents the amino acid sequence translation of SEQ ID NO 41.
[0068] SEQ ID NO 43 represents an artificial nucleotide sequence encoding a TIC1311 protein.
[0069] SEQ ID NO 44 represents the amino acid sequence translation of SEQ ID NO 43.
[0070] SEQ ID NO 45 represents the native Bt nucleotide sequence encoding a TIC1324 protein.
[0071] SEQ ID NO 46 represents the amino acid sequence translation of SEQ ID NO 45.
[0072] SEQ ID NO 47 represents an artificial nucleotide sequence encoding a TIC1324 protein.
[0073] SEQ ID NO 48 represents the amino acid sequence translation of SEQ ID NO 47.
[0074] SEQ ID NO 49 represents the native Bt nucleotide sequence encoding a TIC1407 protein.
[0075] SEQ ID NO 50 represents the amino acid sequence translation of SEQ ID NO 49.
[0076] SEQ ID NO 51 represents an artificial nucleotide sequence encoding a TIC TIC1407 protein.
[0077] SEQ ID NO 52 represents the amino acid sequence translation of SEQ ID NO 51.
[0078] SEQ ID NO 53 represents the native Bt nucleotide sequence encoding a TIC TIC1408 protein.
[0079] SEQ ID NO 54 represents the amino acid sequence translation of SEQ ID NO 53.
[0080] SEQ ID NO 55 represents an artificial nucleotide sequence encoding a TIC1408 protein.
[0081] SEQ ID NO 56 represents the amino acid sequence translation of SEQ ID NO 56.
[0082] SEQ ID NO 57 represents a native Bt nucleotide sequence encoding a TIC1308 protein.
[0083] SEQ ID NO 58 represents an amino acid sequence translation of SEQ ID NO 57.
[0084] SEQ ID NO 59 represents a native Bt nucleotide sequence encoding a TIC1442 protein.
[0085] SEQ ID NO 60 represents an amino acid sequence translation of SEQ ID NO 59.
[0086] SEQ ID NO 61 represents an artificial nucleotide sequence encoding a TIC1308 protein.
[0087] SEQ ID NO 62 represents an amino acid sequence translation of SEQ ID NO 61.
[0088] SEQ ID NO 63 represents an artificial nucleotide sequence encoding a TIC1442 protein.
[0089] SEQ ID NO 64 represents an amino acid sequence translation of SEQ ID NO 63.
DETAILED DESCRIPTION OF THE INVENTION
[0090] The invention relates to methods and compositions for pest control in plants, in particular nematode and/or insect control. In one aspect, the invention relates to controlling, preventing or treating nematode and/or insect infection in transgenic plants. The method comprises, in one embodiment, generation of transgenic plants containing a recombinant construct and expression of such construct to impart such pest resistance to plants. The recombinant construct may comprise a nucleotide sequence encoding one or more proteins, wherein the sequence is operably linked to a heterologous promoter functional in a plant cell, and to cells transformed with the recombinant construct. Cells comprising (meaning including but not limited to) the recombinant construct may be prokaryotic or eukaryotic. In particular, eukaryotic cells may be plant cells. Plants and seeds derived from such transformed plant cells are also contemplated. The transgenic plants or parts thereof of the present invention, in one embodiment, produce one or more pesticidal proteins derived from Bacillus thuringiensis bacterial strains.
[0091] The present invention provides heterologous molecules that are expressed in the cytoplasm of the host cell, or if used in a eukaryotic cell such as a plant cell, may also be directed into the plastid of the plant to provide production of the toxic protein, and including, but not limited to, nucleotide segments that encode polypeptides such as SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, and SEQ ID NO:60 having pesticidal activity. In certain embodiments, the polypeptide having pesticidal activity may share at least about 45%, or at least about 50%, or at least about 51-79%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or 100% sequence identity, to any one or more amino acid sequence(s) set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, or SEQ ID NO:60. The function of the encoded polypeptide may also be determined by measuring the efficacy of the presence of the transgene that encodes it in reducing nematode and/or insect infection, growth, reproduction, or symptomology. For instance, a reduction in root galls, cysts, or worm number of 20% or more, 25% or more, 50% or more, 80% or more, or 95% or more, in a transgenic plant comprising a heterologous nucleotide construct encoding any of the proteins of the present invention, relative to a control plant, for instance an otherwise isogenic plant not comprising the heterologous molecule, under similar conditions, indicates the presence of a functional molecule.
[0092] In certain embodiments, a heterologous molecule provided by the present invention that is directed into the plastid of a plant to provide production of a toxin protein of the present invention may share at least from about 60 to about 79%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, or 100% sequence identity at the nucleotide level with one or more sequence(s) as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63. Thus, in particular embodiments, the heterologous molecule may comprise a sequence encoding a heterologous chloroplast transit peptide.
[0093] Yet another aspect of the invention provides methods for production and for use of one or more of the proteins of the present invention to control nematode and/or insect infestation. Thus, methods for production of a toxin, for instance in a plant cell, are provided. The toxin may then be applied to soil prior to, during, or subsequent to planting of a crop, in order to control or reduce nematode infestation or symptomatology of crop plants grown in that soil.
[0094] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may also be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. The nomenclature for DNA bases as set forth at Title 37 of the United States Code of Federal Regulations, Part 1, section 1.822.
[0095] As used herein, a "transgenic plant" is any plant in which one or more, or all, of the cells of the plant include a transgene. A transgene may be integrated within a nuclear genome or organelle genome, or it may be extra-chromosomally replicating DNA. The term "transgene" means a nucleic acid that is partly or entirely heterologous, foreign, to a transgenic microbe, plant, animal, or cell into which it is introduced. Cells that make up various cell and tissue types of plants include but are not limited to seed, root, leaf, shoot, flower, pollen and ovule.
[0096] "Recombinant DNA" is a polynucleotide having a genetically engineered modification introduced through combination of endogenous and/or exogenous molecules in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit. Recombinant DNA may comprise DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form. An isolated recombinant polynucleotide may exist, for example as a purified molecule, or integrated into a genome, such as a plant cell, or organelle genome or a microbe plasmid or genome. The polynucleotide comprises linked regulatory molecules that cause transcription of an RNA in a plant cell.
[0097] As used herein, "percent identity" means the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, for example nucleotide sequence or amino acid sequence. An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by sequences of the two aligned segments divided by the total number of sequence components in the reference segment over a window of alignment which is the smaller of the full test sequence or the full reference sequence. "Percent identity" ("% identity") is the identity fraction times 100.
[0098] "Expression" means transcription of DNA to produce RNA. The resulting RNA may be without limitation mRNA encoding a protein, antisense RNA, or a double-stranded RNA for use in RNAi technology. Expression also may refer to translation of RNA, i.e. the production of encoded protein from an mRNA.
[0099] As used herein, "promoter" means regulatory DNA molecules for initializing transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. For example it is well known that certain Agrobacterium promoters are functional in plant cells. Thus, plant promoters include promoter DNA obtained from plants, plant viruses (in particular, double stranded DNA viruses) and bacteria such as Agrobacterium and Bradyrhizobium bacteria. Constitutive promoters generally provide transcription in most or all of the cells of a plant. In particular, promoters such as the FMV promoter (FMV, U.S. Pat. No. 6,051,753), the enhanced 35S promoter (E35S, U.S. Pat. No. 5,359,142), rice actin promoter (U.S. Pat. No. 5,641,876), and various chimeric promoters (U.S. Pat. No. 6,660,911) are useful in the present invention. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as "tissue-preferred". Promoters that initiate transcription only in certain tissues are referred to as "tissue specific."
[0100] A number of root-specific or root-enhanced promoters or fragments of such that provide enhanced expression in root tissues relative to other plant tissues have been identified and are known in the art (e.g. U.S. Pat. Nos. 5,110,732, 5,837,848, 5,837,876; 5,633,363; 5,459,252; 5,401,836; 7,196,247; 7,232,940; 7,119,254; and 7,078,589). Examples include root-enhanced or root-specific promoters such as the CaMV-derived as-1 promoter or the wheat PDX1 promoter (U.S. Pat. No. 5,023,179), the acid chitinase gene promoter (Samac et al., Plant Mol. Biol. 25:587-596 (1994); the root specific subdomains of the CaMV35S promoter (Lam et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:7890-7894 (1989); the root-enhanced ORF13 promoter from Agrobacterium rhizogenes (Hansen et al., Mol. Gen. Genet. 254:337-343 (1997); the promoter for the tobacco root-specific gene RB7 (U.S. Pat. No. 5,750,386); and the root cell-specific promoters reported by Conkling et al. (Plant Physiol. 93:1203-1211 (1990). Additional examples include RCc2 and RCc3, promoters that direct root-specific gene transcription in rice (Xu et al., Plant Mol. Biol. 27:237, 1995); soybean root-specific glutamine synthetase promoter (Hire et al., Plant Mol. Biol. 20:207-218, 1992); root-specific control element in the GRP 1.8 gene of French bean (Keller and Baumgartner, Plant Cell 3:1051-1061, 1991.); a root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens (Sanger et al., Plant Mol. Biol. 14:433-443, 1990); and full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean (Miao et al., Plant Cell 3:11-22, 1991). See also Bogusz et al., Plant Cell 2:633-641, 1990, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing non-legume Parasponia andersonii and the related non-nitrogen-fixing non-legume Trema tomentosa are described. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79:69-76). Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al., Plant Mol. Biol. 29(4):759-772, 1995); and rolB promoter (Capana et al., Plant Mol. Biol. 25:681-691, 1994). Examples of nematode-induced promoters include, for instance, the TobRB7 promoter (Opperman et al., Science 263:221-223, 1994), and promoters described in U.S. Pat. Nos. 6,262,344, and 7,193,136.
[0101] The term "resistance," or "tolerance" when used in the context of comparing the effectiveness of a transgene in a transgenic plant, refers to the ability of the transgenic plant to maintain a desirable phenotype when exposed to nematode infestation pressures relative to the phenotype presented by a nematode sensitive non-transgenic plant under similar conditions. The level of resistance can be determined by comparing the physical characteristics of the transgenic plant to non-transgenic plants that either have or have not been exposed to nematode and/or insect infection. Exemplary physical characteristics to observe include plant height, an increase in population of plants that have ability to survive nematode or insect challenge (that is, plants that come in contact with a parasitic nematode or insect may have enhanced root growth, enhanced fruit or grain yield, and decreased reproduction of the nematode or insect infesting the plant or crop, or a decrease in the rate of increase if the pest population). The product of expression of the recombinant DNA may be directly toxic to the nematode (nematicidal) or insect (insecticidal), or may affect the mobility, host finding, feeding site establishment, fecundity or have other nematistatic and/or insectic inhibitory effects.
[0102] "Transformed seed" is the seed which has been generated from the transformed plant. A transformed plant contains transformed cells. A transformed cell is a cell that has been altered by the introduction of an exogenous DNA molecule or in the present invention comprises a heterologous DNA encoding one or more of the proteins of the present invention.
[0103] Pests intended to be within the scope of the present invention include the "lepidopteran pest population" such as Spodoptera frugiperda, Spodoptera exigua, Mamestra configurata, Agrotis ipsilon, Trichoplusia ni, Pseudoplusia includens, Anticarsia gemmatalis, Hypena scabra, Heliothis virescens, Agrotis subterranea, Pseudaletia unipuncta, Agrotis orthogonia, Ostrinia nubilalis, Amyelois transitella Crambus caliginosellus, Herpetogramma licarsisalis, Homoeosoma electellum, Elasmopalpus lignosellu, Cydia pomonella, Endopiza viteana, Grapholita molesta, Suleima helianthana, Plutella xylostella, Pectinophora gossypiella, Lymantria dispar, Blatta orientalis, Blatella asahinai, Blattella germanica, Supella longipalpa, Periplaneta americana, Periplaneta brunnea, Leucophaea maderae, Alabama argillacea, Archips argyrospila, A. rosana, Chilo suppressalis, Cnaphalocrocis medinalis, Crambus caliginosellus, C. teterrellus, Diatraea grandiosella, D. saccharalis, Earias insulana, E. vittella, Helicoverpa armigera, H. zea, Heliothis virescens, Herpetogramma licarsisalis, Lobesia botrana, Pectinophora gossypiella, Phyllocnistis citrella, Pieris brassicae, P. rapae, Plutella xylostella, Spodoptera exigua, S. litura, S. frugiperda, and Tuta absoluta. The "coleopteran pest population" includes Anthonomus grandis, Lissorhoptrus oryzophilu, Sitophilus granaries, Sitophilus oryzae, Hypera punctata, Sphenophorus maidis, Leptinotarsa decemlineata, Diabrotica virgifera virgifera, Diabrotica barberi, Diabrotica undecimpunctata howardi, Chaetocnema pulicaria, Phyllotreta cruciferae, Colaspis brunnea, Oulema melanopus, Zygogramma exclamationis, Epilachna varivestis, Popillia japonica, Cyclocephala boreali, Cyclocephala immaculata, Rhizotrogus majalis, Phyllophaga crinita, Ligyrus gibbosus, Melanotus spp., Conoderus spp., Limonius spp., Agriotes spp., Ctenicera spp., and Aeolus spp., Eleodes spp. The "plant pathogenic nematode population" includes plant parasitic species, for example, Heterodera species, Globodera species, Meloidogyne species, Rotylenchulus species, Hoplolaimus species, Belonolaimus species, Pratylenchus species, Longidorus species, Paratrichodorus species, Ditylenchus species, Xiphinema species, Dolichodorus species, Helicotylenchus species, Radopholus species, Hirschmanniella species, Tylenchorhynchus species, and Trichodorus species, and the like, and specifically includes Heterodera glycines (soybean cyst nematode), Heterodera schachtii (beet cyst nematode), Heterodera avenae, Globodera rostochiensis, Globodera pailida, Pratylenchus zeae (a root knot nematode), Meloidogyne javanica, Pratylenchus brachyurus (a root knot nematode), Meloidogyne hapla, and Meloidogyne incognita.
[0104] The present invention provides recombinant DNA constructs comprising a polynucleotide that, when incorporated in a plant cell, imparts to the plant resistance to nematode and/or insect infection or plant disease caused by such infection (also referred to as infestation). Such constructs also typically comprise a promoter operatively linked to said polynucleotide to provide for expression in the plant cells. Other construct components may include additional regulatory molecules, such as 5' leader regions or 3' untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides. Such recombinant DNA constructs can be assembled using methods known to those of ordinary skill in the art.
[0105] Recombinant constructs prepared in accordance with the present invention also generally include a 3' untranslated DNA region (UTR) that typically contains a polyadenylation sequence following the polynucleotide coding region. Examples of useful 3' UTRs include but are not limited to those from the nopaline synthase gene of Agrobacterium tumefaciens (nos), a gene encoding the small subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and the T7 transcript of Agrobacterium tumefaciens.
[0106] Constructs and vectors may also include a transit peptide for targeting of a protein product, particularly to a chloroplast, leucoplast or other plastid organelle, mitochondria, peroxisome, or vacuole or an extracellular location. For descriptions of the use of chloroplast transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of other such isolated chloroplast proteins include, but are not limited to those associated with the small subunit (SSU) of ribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex protein I and protein II, thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS) and transit peptides described in U.S. Pat. No. 7,193,133. It has been demonstrated in vivo and in vitro that non-chloroplast proteins may be targeted to the chloroplast by use of protein fusions with a heterologous CTP and that the CTP is sufficient to target a protein to the chloroplast. Incorporation of a suitable chloroplast transit peptide, such as, the Arabidopsis thaliana EPSPS CTP (CTP2, Klee et al., Mol. Gen. Genet. 210:437-442, 1987), and the Petunia hybrida EPSPS CTP (CTP4, della-Cioppa et al., Proc. Natl. Acad. Sci. USA 83:6873-6877, 1986) has been show to target heterologous EPSPS protein sequences to chloroplasts in transgenic plants. The production of glyphosate tolerant plants by expression of a fusion protein comprising an amino-terminal CTP with a glyphosate resistant EPSPS enzyme is well known by those skilled in the art, (U.S. Pat. No. 5,627,061, U.S. Pat. No. 5,633,435, U.S. Pat. No. 5,312,910, EP 0218571, EP 189707, EP 508909, and EP 924299). Those skilled in the art will recognize that various chimeric constructs can be made that utilize the functionality of a CTP to import various pesticidal proteins of the present invention into the plant cell plastid.
[0107] Stable methods for plant transformation include virtually any method by which DNA can be introduced into a cell, such as by direct delivery of DNA (for example, by PEG-mediated transformation of protoplasts, by electroporation, by agitation with silicon carbide fibers, and by acceleration of DNA coated particles), by Agrobacterium-mediated transformation, by viral or other vectors. One preferred method of plant transformation is microprojectile bombardment, for example, as illustrated in U.S. Pat. No. 5,015,580 (soy), U.S. Pat. No. 5,550,318 (maize), U.S. Pat. No. 5,538,880 (maize), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No. 6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice) and U.S. Pat. No. 6,399,861 (maize), and U.S. Pat. No. 6,403,865 (maize).
[0108] Detailed procedures for Agrobacterium-mediated transformation of plants, especially crop plants, include, for example, procedures disclosed in U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908, 5,846,797, and 6,624,344 (cotton); U.S. Pat. Nos. 5,416,011, 5,569,834, 5,824,877, 5,914,451 6,384,301, and 7,002,058 (soy); U.S. Pat. Nos. 5,591,616 5,981,840, and 7,060,876 (maize); U.S. Pat. Nos. 5,463,174 and 5,750,871 (Brassica species, including rapeseed and canola), and in U. S. Patent Application Publications 2004/0244075 (maize), 2004/0087030 (cotton) and 2005/0005321 (soybean). Additional procedures for Agrobacterium-mediated transformation are disclosed in WO9506722 (maize). Similar methods have been reported for many plant species, both dicots and monocots, including, among others, peanut (Cheng et al., Plant Cell Rep., 15:653, 1996); asparagus (Bytebier et al., Proc. Natl. Acad. Sci. U.S.A., 84:5345, 1987); barley (Wan and Lemaux, Plant Physiol., 104:37, 1994); rice (Toriyama et al., Bio/Technology, 6:10, 1988; Zhang et al., Plant Cell Rep., 7:379, 1988; wheat (Vasil et al., Bio/Technology, 10:667, 1992; Becker et al., Plant J., 5:299, 1994), alfalfa (Masoud et al., Transgen. Res., 5:313, 1996); Brassica species (Radke et al., Plant Cell Rep., 11:499-505, 1992); and tomato (Sun et al., Plant Cell Physiol., 47:426-431, 2006). Transgenic plant cells and transgenic plants can also be obtained by transformation with other vectors, such as but not limited to viral vectors (for example, tobacco etch virus (TEV), barley stripe mosaic virus (BSMV), and the viruses referenced in Edwardson and Christie, "The Potyvirus Group: Monograph No. 16", 1991, Agric. Exp. Station, Univ. of Florida), plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) or any other suitable cloning vector, when used with an appropriate transformation protocol such as but not limited to bacterial infection (for example, with Agrobacterium as described above), binary bacterial artificial chromosome constructs, direct delivery of DNA (for example, via PEG-mediated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and microprojectile bombardment). It would be clear to one of ordinary skill in the art that various transformation methodologies can be used and modified for production of stable transgenic plants from any number of plant species of interest. For example the construction of stably inherited recombinant DNA constructs and mini-chromosomes can be used as vectors for the construction of transgenic plants (U.S. Pat. No. 7,235,716).
[0109] Plants of the present invention include, but are not limited to, Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro, citrus, clementine, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, forest trees, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, loblolly pine, mango, melon, mushroom, nut, oat, okra, onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, turf, a vine, watermelon, wheat, yams, and zucchini. Crop plants are defined as plants which are cultivated to produce one or more commercial products. Examples of such crops or crop plants include but are not limited to soybean, canola, rape, cotton (cottonseeds), peanut, sunflower, pigeon pea, chickpea, and the like, and grains such as corn, wheat, rice, oat, millet, and rye, and the like. Rape, rapeseed and canola are used synonymously in the present disclosure.
[0110] Transformation methods to provide transgenic plant cells and transgenic plants containing stably integrated recombinant DNA are preferably practiced in tissue culture on media and in a controlled environment. Recipient cell targets include but are not limited to meristem cells, callus, immature embryos or parts of embryos, gametic cells such as microspores, pollen, sperm, and egg cells. Any cell from which a fertile plant can be regenerated is contemplated as a useful recipient cell for practice of the invention. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells which are capable of proliferating as callus can serve as recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention (for example, various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U. S. Patent Application Publication 2004/0216189.
[0111] In general transformation practice, DNA is introduced into only a small percentage of target cells in any one transformation experiment. Marker genes are generally used to provide an efficient system for identification of those cells that are transformed by a transgenic DNA construct. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the antibiotics or herbicides to which a plant cell may be resistant can be a useful agent for selection. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is expressed at sufficient levels to permit cell survival in the presence of the selective agent. Cells can be tested further to confirm integration of the recombinant DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin or paromomycin (val), hygromycin B (aph IV), gentamycin (aac3 and aacC4) and glufosinate (bar or pat), glyphosate (EPSPS), and dicamba (dicamba monooxygenase). Examples of useful selective marker genes and selection agents are illustrated in U.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and 6,118,047. Screenable markers or reporters, such as markers that provide an ability to visually identify transformants can also be employed. Non-limiting examples of useful screenable markers include, for example, a gene expressing a protein that produces a detectable color by acting on a chromogenic substrate (for example, beta-glucuronidase, GUS, uidA, or luciferase, luc) or that itself is detectable, such as green fluorescent protein (GFP, gfp) or an immunogenic molecule. Those of skill in the art will recognize that many other useful markers or reporters are available for use.
[0112] The recombinant DNA constructs of the invention can be stacked with other recombinant DNA for imparting additional agronomic traits (such as in the case of transformed plants, traits including but not limited to herbicide resistance, insect resistance, cold germination tolerance, water deficit tolerance, enhanced yield, enhanced quality, fungal, viral, and bacterial disease resistance) for example, by expressing other transgenes. The recombinant DNA constructs of the present invention can also be transformed into plant varieties that carry natural pest or pathogen resistance genes to enhance the efficacy of the resistance phenotype. Constructs for coordinated decrease and/or increase of gene expression are disclosed in U.S. Patent Application Publication 2004/0126845 A1. Seeds of transgenic, fertile plants can be harvested and used to grow progeny generations, including hybrid generations, of transgenic plants of this invention that include the recombinant DNA construct in their genome. Thus, in addition to direct transformation of a plant with a recombinant DNA construct of this invention, transgenic plants of the invention can be prepared by crossing a first plant having the recombinant DNA with a second plant lacking the construct. For example, the recombinant DNA can be introduced into a plant line that is amenable to transformation to produce a transgenic plant, which can be crossed with a second plant line to introgress the recombinant DNA into the resulting progeny. A transgenic plant of the invention can be crossed with a plant line having other recombinant DNA or naturally occurring genetic regions that confers one or more additional trait(s) (such as, but not limited to, herbicide resistance, pest or disease resistance, environmental stress resistance, modified nutrient content, and yield improvement) to produce progeny plants having recombinant DNA that confers both the desired target sequence expression behavior and the additional trait(s). Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross segregate such that some of the plant will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA. Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, for example, usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as one original transgenic parental line but for the recombinant DNA of the other transgenic parental line.
[0113] Other proteins and toxic agents can be used together with one or more proteins of the present invention to control plant pathogenic nematode and/or insect infestation and to reduce the likelihood of development of resistance to any single method of control. Such other proteins and toxic agents include but are not limited to, as applicable to either nematode or insect control, methylketone synthase, dsRNA expressed in the cell and targeting for suppression one or more essential, housekeeping, reproductive or developmental gene, other proteins that are known in the art to be toxic to plant pathogenic nematodes or insects such as Cry and VIP proteins (lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/index.html" on the world wide web, which is properly referenced as Crickmore et. al. (2010) "Bacillus thuringiensis toxin nomenclature"), as well as chemical nematicides used in seed treatments or soil drenches. Topically applied dsRNA methods are also known in the art that can be applied to a plant expressing one or more of the proteins of the present invention. Such topical applications can be effective in causing a systemic effect in the plant that result in nematode or insect control by applying to the plant a dsRNA molecule that targets for regulation a gene in the plant involved in such resistance. All such combinations are within the scope of the present invention.
[0114] The transgenic plant, plant part, seed or progeny plants of the present invention can be processed into products useful in commerce. These products, commodity products, include but are not limited to meal, flour, oil, hay, starch, juice, protein extract, and fiber.
[0115] The proteins of the present invention have been identified using a variety of methods. One method has been to identify previously known Bt proteins that exhibit a mass less than about 40 kDa, or less than about 35 kDa, or less than about 30 kDa, or less than about 28 kDa, or less than about 25 kDa, or less than about 20 kDa, or less than about 15 kDa. Such proteins include but are not limited to the smaller component of most known binary Bt toxins, such as Cry34/35 (PS149B1), TIC100/101, ET33/34, ET80/76, and the like. Other proteins known in the art include TIC901, TIC1201, TIC407, TIC417, TIC431, ET70, VIP proteins such as VIP3Aa and the like, which are all generally small toxin proteins that are known to exhibit insecticidal activity. The inventors herein have identified that such smaller toxin molecules, when provided in the diet of a C. elegans nematode, exhibit various levels of inhibitory effects. Surprisingly, it has also been observed that the nematidical activity of these proteins can be imparted through the diet of a cyst nematode more effectively by truncating the proteins to smaller sizes, whether truncated at the C terminus, the N terminus, or both. Truncated versions typically exhibit a mass of from about 14 to about 28-30 kDa, and exhibit improved bioactivity likely because the ability of the cyst nematode to uptake proteins larger than about 30 kDa is limited (Urwin et al. ((1997) Plant J. 12:455) and Bockenhoff & Grundler ((1994) Parasitology 109:249). TIC1501 (about 27 kDa), TIC1503 (about 34 kDa), and TIC1506 (about 36 kDa) represent various fragments of the TIC1201 protein, 1201 being previously shown to exhibit coleopteran toxic effects. Surprisingly, the truncated versions less than 36 kDa exhibit significant nematicidal effects.
[0116] Proteins of the present invention have also been identified de novo, and these include the proteins listed herein as TIC614, TIC615, TIC1277, TIC1278, TIC1308, TIC1310, TIC1311, TIC1324, TIC1407, TIC1408, and TIC1442. Such proteins were identified by various methods, whether being directly amplified from various Bt strain genomes, or identified by high throughput sequence analysis of various Bt genomes. In either case, genomic DNA segments are obtained and analyzed using bioinformatic techniques that result in the identification of all or portions of open reading frames encoding protein segments. The resulting protein segments are then characterized versus all known protein sequences in the art, and to the extent that there is any similarity to a toxin molecule, the complete sequence of the open reading frame encoding the protein is obtained. Proteins that are identified that exhibit a mass of less than about 40 kDa, or preferably less than about 30 kDa are then evaluated in a C. elegans assay to determine if any effects are observed relative to C. elegans survival. Toxins exhibiting nematicidal properties are then evaluated for other pesticidal properties, particularly insecticidal activity. Surprisingly, the above referenced proteins all exhibited nematicidal activity, and some exhibited insecticidal activity as reported in the examples below.
EXAMPLES
[0117] The following examples are illustrative of the invention, which may be embodied in various forms and are not to be interpreted as limiting the scope or content of the disclosure in any way.
Example 1
DNA Molecules Encoding Bt Toxin Proteins
[0118] Toxin ET34 (SEQ ID NO:1) has been previously described (U.S. Pat. No. 6,063,756). A secretion signal from the gene P139 (First 75 nucleotide of SEQ ID NO:1 from the WIPO Publication Number WO9408010) was operably linked to the 5' end of the ET34 (SEQ ID NO:5) to enable its secretion outside the plasma membrane to avoid potential toxicity to the plant cell and to allow easy access of the protein to the pest.
[0119] TIC1506 (SEQ ID NO:9), TIC1501 (SEQ ID NO:13), and TIC 1503 (SEQ ID NO:17) are Bt nucleotide of various fragments of TIC1201 which is 364 amino acids long as set forth in SEQ ID NO 6 of US Patent Application Publication Number US2006-0191034 A1. TIC1506 is 321 amino acids long without the putative N terminal signal peptide of TIC1201 and contains amino acid 44 to 364 of TIC1201 with a methionine residue substituted for the native alanine residue at amino acid position 44. TIC1501 is 227 amino acids long without the putative N terminal signal peptide and a portion of the C terminal of TIC1201 and contains amino acids 44 to 270 of TIC1201 with a methionine residue substituted for the native alanine residue at position 44. TIC1503 is 301 amino acids long without the putative N terminal signal peptide and a portion of the C terminal of TIC1201 and contains amino acids 44 to 344 of TIC1201 with a methionine residue substituted for the native alanine residue at position 44.
[0120] Proteins exhibiting pesticidal properties have been identified in various Bt strains. Open reading frames encoding the amino acid sequences, TIC614 (SEQ ID NO: 22), TIC615 (SEQ ID NO: 26), TIC1277 (SEQ ID NO: 30), TIC1278 (SEQ ID NO: 34), TIC1310 (SEQ ID NO: 38), TIC1311 (SEQ ID NO: 42), TIC1324 (SEQ ID NO: 46), TIC1407(SEQ ID NO: 50), and TIC1408 (SEQ ID NO: 54), exhibiting various degrees of homology to previously known Bt toxin segments were identified. Complete forward and reverse sequence analysis of such open reading frames resulted in the identification of deduced amino acid compositions that exhibit the size and potential for pesticidal (nematicidal and/or insecticidal) activity.
[0121] Proteins exhibiting pesticidal properties have been identified in various Bt strains. Open reading frames encoding the amino acid sequences, TIC1308 (SEQ ID NO: 58) and TIC1442 (SEQ ID NO: 60), exhibiting various degrees of homology to previously known Bt toxin segments were identified. Complete forward and reverse sequence analysis of such open reading frames resulted in the identification of deduced amino acid compositions that exhibit the size and potential for pesticidal (nematicidal and/or insecticidal) activity.
Example 2
Expression of Pesticidal Polypeptides from Polynucleotides
[0122] Open reading frames of ET34 (SEQ ID NO: 1), P139-ET34 (SEQ ID NO: 5), TIC1506 (SEQ ID NO:9), TIC1501 (SEQ ID NO:13), TIC 1503 (SEQ ID NO:17), TIC614 (SEQ ID NO:21), TIC615 (SEQ ID NO:25), TIC1277 (SEQ ID NO:29), TIC1278 (SEQ ID NO:33), TIC1310 (SEQ ID NO:37), TIC1311 (SEQ ID NO:41), TIC1324 (SEQ ID NO:45), TIC1407 (SEQ ID NO:49), TIC1408 (SEQ ID NO:53), TIC1308 (SEQ ID NO: 57), and TIC1442 (SEQ ID NO: 59) encoding the deduced amino acid compositions exemplified in Example 1 were cloned into a Bt/E. coli shuttle plasmid enabling the expression of the deduced amino acid composition in either an acrystalliferous Bt strain or in an E. coli bacterium. Recombinant plasmids were transformed into an acrystalliferous Bt expression host after confirming the DNA sequence of the polynucleotide encoding the polypeptide. The gene of interest was cloned downstream of either a Bacillus vegetative stage or sporulation stage specific promoter to allow the protein to be expressed respectively during vegetative growth or during sporulation of the recombinant Bt strain. Conditions for vegetative expression of the protein included growing the cells for 24-48 hrs in Terrific Broth medium at 25-28.degree. C. Crystal formation is one characteristic of certain Bt toxin proteins. Proteins that were confirmed to produce crystals when expressed in the acrystalliferous Bt strain were further evaluated. Certain proteins accumulated in the cells and/or were secreted into the culture medium. Both the cell pellets and the culture were analyzed by SDS-PAGE for expression of the expected protein. Conditions for expression from a sporulation specific promoter included growing the cells for 96 hrs at 25-28.degree. C. in C2 medium. Protein crystals were formed during sporulation and released from lysed cells as sporulation was completed. Spores and crystals were collected by centrifugation at 4000.times.g for 20 minutes, resuspended in wash buffer (10 mM Tris, 0.1 mM EDTA and 0.005% Triton X100 pH 6.8) and collected again by centrifugation. The spore-crystals pellets were then resuspended in 1/10.sup.th of the original culture volume. The 10.times. concentrated spore-crystal preparation were analyzed by SDS-PAGE the presence of the expected protein.
Example 3
Nematode and Insect Bioassays
[0123] C. elegans feeding screens have been successfully used to identify plant pathogenic nematode-active toxins, for example for SCN and RKN from the order Tylenchida (Wei et al., 2003, PNAS, USA, 100: 2760). The proteins of the present invention were expressed and provided in the diet of a C. elegans nematode, essentially following the method of Wei et al. Efficacy was scored on a scale of 1-3, where a score of 1 represents normal health and reproduction of C. elegans, and a score of 3 represents no reproduction or poor health of C. elegans. Toxins ET34 (SEQ ID NO: 2), TIC1501 (SEQ ID NO: 14), TIC1503 (SEQ ID NO: 18), TIC614 (SEQ ID NO:22), TIC615 (SEQ ID NO:26), TIC1277 (SEQ ID NO:30), TIC1278 (SEQ ID NO:34), TIC1310 (SEQ ID NO:38), TIC1311 (SEQ ID NO:42), TIC1324 (SEQ ID NO:46), and TIC1407(SEQ ID NO:50), each exhibited a score of 3 where as TIC1408 (SEQ ID NO:54) exhibited a score of 2.75. Proteins TIC1308 (SEQ ID NO: 58) and TIC1442 (SEQ ID NO: 60), each exhibited a score of 1. Proteins were expressed from a vegetative specific promoter and fed to insects by applying sporulated bacterial cells or culture supernatant to artificial insect diet. For polypeptides expressed from a sporulation specific promoter a 10-20.times. spore-crystal preparation with about 500-4000 ppm protein was applied to the insect diet. Stunting or mortality was observed on one or more of these insects: CEW (Corn Ear Worm); SCR (Southern corn Root worm); WCR (western corn root worm); ECB (European corn borer); WTPB (Western tarnished plant bug); TPB (Tarnished plant bug); FAW (Fall army worm); CPB (Colorado potato beetle). TIC1277 and TIC1311 were found to cause significant stunting of ECB and TIC1310 was found to cause significant stunting of WTPB and significant stunting and mortality of CPB.
Example 4
Transformed Plants
[0124] Nucleotide segments encoding TIC1506 (SEQ ID NO:11), TIC1503(SEQ ID NO: 19), TIC614 (SEQ ID NO:3), TIC1324 (SEQ ID NO:27), TIC1407(SEQ ID NO:31), TIC1408 (SEQ ID NO:35), are codon-optimized for plant expression and operably linked to one or more plant functional promoters and introduced into plant cells. Recombinant plants are regenerated from such transformed plant cells, and the regenerated plants are evaluated for resistance to pest infestation, such as insect tolerance and/or plant pathogenic nematode tolerance.
[0125] Nucleotide segments encoding ET34 (SEQ ID NO:3), ET34+P139 secretion signal (SEQ ID NO:7), TIC1501 (SEQ ID NO:15), TIC615 (SEQ ID NO:7), TIC1277 (SEQ ID NO:11), TIC1278 (SEQ ID NO:15), TIC1310 (SEQ ID NO:19), TIC1311 (SEQ ID NO:23), TIC1308 (SEQ ID NO: 61), and TIC1442 (SEQ ID NO: 63) were codon-optimized for plant expression and operably linked to one or more plant functional promoters and introduced into plant cells. Recombinant plants were regenerated from such transformed plant cells, and the regenerated plants were evaluated for resistance to pest infestation, such as insect tolerance and/or plant pathogenic nematode tolerance.
Example 5
Transformation of Soybean
[0126] This example describes a method of producing transgenic soybean plants and transgenic plant parts such as seeds. Other methods are known in the art of plant cell transformation that can be applied to transform plant cells and regenerate transgenic plants using the recombinant constructs of the invention. The methods of obtaining transgenic soybean plants and seeds are used as essentially disclosed in US Patent Application Publication Number US2009-0138985A1. Briefly, Agrobacterium containing a construct of Example 5 are grown in Luria Burtani (LB) media containing spectinomycin at about 28.degree. C. for over night. The bacterial culture is centrifuged, pellet washed, and resuspended in INO medium for inoculating wet or dry mature embryos explants. The explants are mixed with the Agrobacterium cell suspensions and briefly exposed to sonication energy from a standard laboratory water bath cleaning sonicator. The explants are drained of any liquid and transferred to containers containing filter paper moistened with INO media and co-cultured in a lighted chamber at about 16 hours of light (5 uE) at about 23.degree. to 28 C..degree. for 1 to 5 days. After co-culture, the explants are placed directly onto regeneration media containing a selective agent such as spectinomycin from about 7 to about 42 days. The cultures are subsequently transferred to a media suitable for the recovery of transformed plantlets. Spectinomycin resistant shoots that have green buds or leaves are considered transformed and placed in soil or on a soil substitute for rooting in the presence or absence of the selective agent. Progeny transgenic plants and seed are selected that provide pest resistance, especially nematode resistance.
Example 6
Testing of Transgenic Plant for Soybean Cyst Nematode (SCN) Resistance
[0127] An SCN pot assay was used to evaluate the resistance of transgenic soybean plants comprising one or more of the polynucleotide sequences of SEQ ID NOs: 3, 7, and 15 to infection by and reproduction of the SCN (Heterodera glycines) on roots. Three or four inch diameter square pots were filled with clean sand and watered thoroughly. Transgenic and control soybean seeds, or alternatively any rooted plant parts, were planted one per pot in the center of the pot and watered well to remove air pockets. The pots were incubated in the greenhouse or growth chamber at 20.degree. C. to 30.degree. C. until the plants reached a suitable age for inoculation. Soybeans started from seed were typically inoculated 2-3 weeks after planting, while transplants were inoculated 1-3 days after planting. The test inoculum consisted of eggs from ripe H. glycines cysts collected from the soil and roots of infested soybean plants. An 80 micron mesh sieve was used to collect the cysts, which were then crushed in a Tenbroeck glass tissue homogenizer to release the eggs. The eggs were further purified by sieving and centrifugation over 40 percent sucrose solution at 4000 RPM for 5 minutes. Inoculum for an experiment consisted of water containing 500 vermiform eggs per mL. Five mL of the egg suspension was applied over the surface of the sand containing the test plants and the eggs were lightly watered in. The test plants were then returned to the greenhouse or growth chamber and incubated for 3-4 weeks to allow for root infection and cyst formation. The roots were then harvested. The severity of nematode infection was measured by counting the number of nematode cysts adhering to the root system.
[0128] Transgenic soybean plants comprising SEQ ID NO: 3 were tested in six different constructs, where in each construct SEQ ID NO: 3 was operably linked to a different promoter. Transgenic soybean plants comprising SEQ ID NO: 7 were tested in two different constructs, each construct having a different promoter. Transgenic soybean plants comprising SEQ ID NO: 15 were tested expressed from one construct.
[0129] Table 1 reports data illustrating plants from multiple events per multiple constructs that were evaluated for and determined to have significant cyst reduction against SCN when compared to the untransformed soybean cultivar. The number of plant roots tested was about equally distributed among the number of events tested.
TABLE-US-00001 TABLE 1 Severity of Soybean Cyst Nematode cyst infection on soybean plant roots Number Number of SEQ of plant Result (compared to Protein ID Construct Events roots control non-transgenic Name NO: Name tested tested plants) ET34 3 128213 14 177 1 of 14 events showed 35.4% cyst reduction ET34 3 126168 15 389 7 of 15 events showed more than 46.7% cyst reduction ET34 3 126626 15 191 0 of 15 events showed significant cyst reduction ET34 3 126630 16 204 1 of 16 events showed 42.0% cyst reduction ET34 3 127056 15 191 3 of 15 events showed more than 41.5% cyst reduction ET34 3 128296 14 168 2 of 14 events showed more than 33% cyst reduction P139 + 7 126169 15 176 0 of 15 events showed ET34 the significant cyst reduction P139 + 7 126628 16 192 0 of 16 events showed ET34 the significant cyst reduction TIC1501 15 133535 5 18 3 of 5 events showed more than 45% cyst reduction
Example 7
Testing of Transgenic Arabidopsis Plant for Beet Cyst Nematode (BCN) Resistance
[0130] Transgenic Arabidopsis seeds and plants comprising one or more of the polynucleotide sequences of SEQ ID NOs 3, 15, 27, 31, 35, 39, 43, 61, and 63 were produced by the method of Clough et al., 1998 (Plant J. 16:735-743) and tested for Beet Cyst Nematode (BCN) resistance by the method of Sijmons et al., 1991 (Plant J. 1: 245-254) and Vaghchhipawala et al., 2004 (Genome 47: 404-13).
[0131] Arabidopsis (variety Columbia-0) seeds were surface sterilized and rinsed with sterile water and plated on B5 medium. Plates were incubated at 23-25.degree. C. with a 16 hour light/8 hour dark cycle for 7-10 days. BCN eggs were placed on the sterile filter paper and hatched in 5 mM ZnSO4 solution for 5-7 days at 25.degree. C. J2 stage juvenile nematodes were collected, rinsed in sterile water, and treated with 0.5% chlorhexidine diacetate for 10-15 minutes. Treated juvenile J2 nematodes were collected and rinsed twice in sterile water and stored in sterile water for infestation purposes.
[0132] For the infestation assay, about 10-15 Arabidopsis seeds were sprinkled on steamed sand in a pot and covered with a clear plastic dome. Several such dome/flat combos were placed in a flat and then covered with a black tray and transferred to a cold room for vernalization. On day 4, the flat was taken out of the cold room, the black tray is removed, and the flat was placed in a growth chamber for acclimating seeds at 26.degree. C., 70% humidity, 140-180 .mu.E light, 12 hours day length. The pots were watered and fertilized as needed. Three weeks after planting, the Arabidopsis plants were inoculated with 3,000 BCN eggs. About 35 days after inoculation the plants were harvested and cysts extracted by washing the plant's roots in a bucket of water and filtering the water through a 16 mesh sieve on top of a 50 mesh sieve. The cysts were collected off the top of the 50 mesh sieve and counted. Plants with lesser number of cysts compared to non-transgenic or transgenic control were considered resistant to BCN.
[0133] Table 2 reports data illustrating plants from multiple events per multiple constructs that were evaluated for and determined to have lesser number of BCN cysts compared to the untransformed Arabidopsis parental background. The number of plant roots tested was nearly equally distributed among the number of events tested.
TABLE-US-00002 TABLE 2 Severity of Beet Cyst Nematode cyst infection on Arabidopsis thaliana plant roots Number Resulting number Number of of events having a SEQ of plant lower mean cyst count Protein ID Construct Events roots compared to non- Name NO: Name tested tested transgenic control ET34 3 140057 6 72 3 TIC1501 15 140056 6 72 3 TIC615 27 139822 6 72 4 TIC1277 31 142259 6 71 2 TIC1278 35 141647 6 72 4 TIC1310 39 142255 6 72 1 TIC1311 43 141644 6 71 6 TIC1308 61 141250 6 70 2 TIC1422 63 141205 6 72 2
[0134] Various patent and non-patent publications are cited herein, the disclosures of each of which are incorporated herein by reference in their entireties. Documents cited herein as being available from the World Wide Web at certain internet addresses are also incorporated herein by reference in their entireties.
[0135] As various modifications could be made in the compositions and methods herein described and illustrated without departing from the scope of the invention, it is intended that all matter contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative rather than limiting. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims appended hereto and their equivalents.
Sequence CWU
1
1
641381DNABacillus thuringiensis 1atgacagtat ataacgcaac tttcaccatt
aatttctata atgaaggaga atgggggggg 60ccagaaccat atggttatat aaaagcatat
cttacaaatc cagatcatga ttttgaaatt 120tggaaacaag atgattgggg gaaaagtact
cctgagagaa gtacttatac gcaaacgatt 180aaaataagta gcgacactgg ttcccctata
aaccaaatgt gtttttatgg tgatgtgaaa 240gaatacgacg taggaaatgc agatgatatt
ctcgcttatc caagtcaaaa agtatgcagt 300acacctggtg taacagtacg acttgatggc
gatgagaaag gttcttatgt gacaattaag 360tattccttga ctccagcata a
3812126PRTBacillus thuringiensis 2Met
Thr Val Tyr Asn Ala Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly 1
5 10 15 Glu Trp Gly Gly Pro Glu
Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr 20
25 30 Asn Pro Asp His Asp Phe Glu Ile Trp Lys
Gln Asp Asp Trp Gly Lys 35 40
45 Ser Thr Pro Glu Arg Ser Thr Tyr Thr Gln Thr Ile Lys Ile
Ser Ser 50 55 60
Asp Thr Gly Ser Pro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 65
70 75 80 Glu Tyr Asp Val Gly
Asn Ala Asp Asp Ile Leu Ala Tyr Pro Ser Gln 85
90 95 Lys Val Cys Ser Thr Pro Gly Val Thr Val
Arg Leu Asp Gly Asp Glu 100 105
110 Lys Gly Ser Tyr Val Thr Ile Lys Tyr Ser Leu Thr Pro Ala
115 120 125 3381DNAArtificial
sequencerecombinant polynucleotide encoding an ET34 protein.
3atgactgttt acaacgctac tttcactatt aacttctaca acgagggtga gtggggtgga
60cctgagcctt acggttacat taaggcttac ttgactaatc cagatcatga tttcgagatt
120tggaagcaag atgattgggg caagagcact ccagagcgct ctacctacac ccaaaccatt
180aagatttcat ccgataccgg atcacccatc aatcagatgt gcttctacgg agatgttaag
240gagtatgatg ttggcaatgc tgacgacatc cttgcctatc cctcccagaa ggtctgtagt
300acacccggcg tcacagtgag gctcgacggc gacgagaagg gttcctatgt aaccatcaag
360tattcgctga cgcctgcatg a
3814126PRTArtificial sequencean amino acid sequence translation of SEQ ID
NO 3 from nucleotide position 1 through nucleotide position 378 4Met
Thr Val Tyr Asn Ala Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly 1
5 10 15 Glu Trp Gly Gly Pro Glu
Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr 20
25 30 Asn Pro Asp His Asp Phe Glu Ile Trp Lys
Gln Asp Asp Trp Gly Lys 35 40
45 Ser Thr Pro Glu Arg Ser Thr Tyr Thr Gln Thr Ile Lys Ile
Ser Ser 50 55 60
Asp Thr Gly Ser Pro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 65
70 75 80 Glu Tyr Asp Val Gly
Asn Ala Asp Asp Ile Leu Ala Tyr Pro Ser Gln 85
90 95 Lys Val Cys Ser Thr Pro Gly Val Thr Val
Arg Leu Asp Gly Asp Glu 100 105
110 Lys Gly Ser Tyr Val Thr Ile Lys Tyr Ser Leu Thr Pro Ala
115 120 125 5453DNAArtificial
sequencerecombinant polynucleotide encoding a P139 secretion signal
peptide (nucleotide position 1-75) fused in frame to a native Bt
nucleotide sequence encoding a ET34 protein (nucleotide position
76-450) 5atgaggagtt tcgccgtctt gttgccgctt ctcgtcacct tctgcgtggt
ggcgcctcct 60tccgacgccg ccaccacagt atataacgca actttcacca ttaatttcta
taatgaagga 120gaatgggggg ggccagaacc atatggttat ataaaagcat atcttacaaa
tccagatcat 180gattttgaaa tttggaaaca agatgattgg gggaaaagta ctcctgagag
aagtacttat 240acgcaaacga ttaaaataag tagcgacact ggttccccta taaaccaaat
gtgtttttat 300ggtgatgtga aagaatacga cgtaggaaat gcagatgata ttctcgctta
tccaagtcaa 360aaagtatgca gtacacctgg tgtaacagta cgacttgatg gcgatgagaa
aggttcttat 420gtgacaatta agtattcctt gactccagca taa
4536150PRTArtificial sequencean amino acid sequence
translation of SEQ ID NO 5. 6Met Arg Ser Phe Ala Val Leu Leu Pro Leu
Leu Val Thr Phe Cys Val 1 5 10
15 Val Ala Pro Pro Ser Asp Ala Ala Thr Thr Val Tyr Asn Ala Thr
Phe 20 25 30 Thr
Ile Asn Phe Tyr Asn Glu Gly Glu Trp Gly Gly Pro Glu Pro Tyr 35
40 45 Gly Tyr Ile Lys Ala Tyr
Leu Thr Asn Pro Asp His Asp Phe Glu Ile 50 55
60 Trp Lys Gln Asp Asp Trp Gly Lys Ser Thr Pro
Glu Arg Ser Thr Tyr 65 70 75
80 Thr Gln Thr Ile Lys Ile Ser Ser Asp Thr Gly Ser Pro Ile Asn Gln
85 90 95 Met Cys
Phe Tyr Gly Asp Val Lys Glu Tyr Asp Val Gly Asn Ala Asp 100
105 110 Asp Ile Leu Ala Tyr Pro Ser
Gln Lys Val Cys Ser Thr Pro Gly Val 115 120
125 Thr Val Arg Leu Asp Gly Asp Glu Lys Gly Ser Tyr
Val Thr Ile Lys 130 135 140
Tyr Ser Leu Thr Pro Ala 145 150 7453DNAArtificial
sequencea recombinant polynucleotide sequence encoding a P139
secretion signal peptide (nucleotide position 1-75) fused in frame
to a synthetic nucleotide sequence encoding a ET34 protein
(nucleotide position 76-450) 7atgaggagtt tcgccgtctt gttgccgctt ctcgtcacct
tctgcgtggt ggcgcctcct 60tccgacgccg ccaccactgt ttacaacgct actttcacta
ttaacttcta caacgagggt 120gagtggggtg gacctgagcc ttacggttac attaaggctt
acttgactaa tccagatcat 180gatttcgaga tttggaagca agatgattgg ggcaagagca
ctccagagcg ctctacctac 240acccaaacca ttaagatttc atccgatacc ggatcaccca
tcaatcagat gtgcttctac 300ggagatgtta aggagtatga tgttggcaat gctgacgaca
tccttgccta tccctcccag 360aaggtctgta gtacacccgg cgtcacagtg aggctcgacg
gcgacgagaa gggttcctat 420gtaaccatca agtattcgct gacgcctgca tga
4538150PRTArtificial sequencean amino acid
sequence translation of SEQ ID NO 7. 8Met Arg Ser Phe Ala Val Leu
Leu Pro Leu Leu Val Thr Phe Cys Val 1 5
10 15 Val Ala Pro Pro Ser Asp Ala Ala Thr Thr Val
Tyr Asn Ala Thr Phe 20 25
30 Thr Ile Asn Phe Tyr Asn Glu Gly Glu Trp Gly Gly Pro Glu Pro
Tyr 35 40 45 Gly
Tyr Ile Lys Ala Tyr Leu Thr Asn Pro Asp His Asp Phe Glu Ile 50
55 60 Trp Lys Gln Asp Asp Trp
Gly Lys Ser Thr Pro Glu Arg Ser Thr Tyr 65 70
75 80 Thr Gln Thr Ile Lys Ile Ser Ser Asp Thr Gly
Ser Pro Ile Asn Gln 85 90
95 Met Cys Phe Tyr Gly Asp Val Lys Glu Tyr Asp Val Gly Asn Ala Asp
100 105 110 Asp Ile
Leu Ala Tyr Pro Ser Gln Lys Val Cys Ser Thr Pro Gly Val 115
120 125 Thr Val Arg Leu Asp Gly Asp
Glu Lys Gly Ser Tyr Val Thr Ile Lys 130 135
140 Tyr Ser Leu Thr Pro Ala 145
150 9966DNABacillus thuringiensis 9gcaataactc catatgctga atcttatatt
gatactgttc aagatagaat gaaacaaaga 60gatagggaat caaaactaac tggtaaacca
ataaatatgc aagaacaaat aatagatgga 120tggtttttag ctagattctg gatatttaaa
gatcaaaata acaatcatca aacaaataga 180tttatatcct ggtttaaaga taatcttgct
agttcgaagg ggtatgacag tatagcagaa 240caaatgggct taaaaataga agcattaaat
gatatggatg taacaaatat tgattataca 300tctaaaacag gtgataccat atataatgga
atttctgaac taacaaatta tacaggaaca 360acccaaaaaa tgaaaaccga tagttttcaa
agagattata caaaatctga atccacttca 420gtaacaaatg ggttacaatt aggatttaaa
gttgctgcta agggagtagt tgcattagca 480ggtgcagatt ttgaaacaag tgttacctat
aatttatcat ctactacaac tgaaacaaat 540acaatatcgg ataagtttac tgttccatct
caagaagtta cattatcccc aggacataaa 600gcagtggtga aacatgattt gagaaaaatg
gtgtattttg ggactcatga tttaaagggt 660gatttaaaag taggttttaa tgataaagag
attgtacaaa aatttattta tccaaattat 720agatcaattg atttatctga tattcgtaaa
acaatgattg aaattgataa atggaatcat 780gtaaatacca ttgactttta tcaattagtt
ggagttaaaa atcatataaa aaatggtgat 840actttatata tagatacccc ggccgaattt
acatttaatg gagctaatcc atattataga 900gcaacattta cagaatacga cgagaacgga
aatcctgttc aaacaaagat tttaagtgga 960aattaa
96610321PRTArtificial sequencean amino
acid sequence translation of SEQ ID NO 9. 10Met Ile Thr Pro Tyr Ala
Glu Ser Tyr Ile Asp Thr Val Gln Asp Arg 1 5
10 15 Met Lys Gln Arg Asp Arg Glu Ser Lys Leu Thr
Gly Lys Pro Ile Asn 20 25
30 Met Gln Glu Gln Ile Ile Asp Gly Trp Phe Leu Ala Arg Phe Trp
Ile 35 40 45 Phe
Lys Asp Gln Asn Asn Asn His Gln Thr Asn Arg Phe Ile Ser Trp 50
55 60 Phe Lys Asp Asn Leu Ala
Ser Ser Lys Gly Tyr Asp Ser Ile Ala Glu 65 70
75 80 Gln Met Gly Leu Lys Ile Glu Ala Leu Asn Asp
Met Asp Val Thr Asn 85 90
95 Ile Asp Tyr Thr Ser Lys Thr Gly Asp Thr Ile Tyr Asn Gly Ile Ser
100 105 110 Glu Leu
Thr Asn Tyr Thr Gly Thr Thr Gln Lys Met Lys Thr Asp Ser 115
120 125 Phe Gln Arg Asp Tyr Thr Lys
Ser Glu Ser Thr Ser Val Thr Asn Gly 130 135
140 Leu Gln Leu Gly Phe Lys Val Ala Ala Lys Gly Val
Val Ala Leu Ala 145 150 155
160 Gly Ala Asp Phe Glu Thr Ser Val Thr Tyr Asn Leu Ser Ser Thr Thr
165 170 175 Thr Glu Thr
Asn Thr Ile Ser Asp Lys Phe Thr Val Pro Ser Gln Glu 180
185 190 Val Thr Leu Ser Pro Gly His Lys
Ala Val Val Lys His Asp Leu Arg 195 200
205 Lys Met Val Tyr Phe Gly Thr His Asp Leu Lys Gly Asp
Leu Lys Val 210 215 220
Gly Phe Asn Asp Lys Glu Ile Val Gln Lys Phe Ile Tyr Pro Asn Tyr 225
230 235 240 Arg Ser Ile Asp
Leu Ser Asp Ile Arg Lys Thr Met Ile Glu Ile Asp 245
250 255 Lys Trp Asn His Val Asn Thr Ile Asp
Phe Tyr Gln Leu Val Gly Val 260 265
270 Lys Asn His Ile Lys Asn Gly Asp Thr Leu Tyr Ile Asp Thr
Pro Ala 275 280 285
Glu Phe Thr Phe Asn Gly Ala Asn Pro Tyr Tyr Arg Ala Thr Phe Thr 290
295 300 Glu Tyr Asp Glu Asn
Gly Asn Pro Val Gln Thr Lys Ile Leu Ser Gly 305 310
315 320 Asn 11966DNAArtificial
sequencerecombinant polynucleotide encoding a TIC1506 protein.
11atgattactc cttacgctga gtcttacatt gatactgttc aagatcgcat gaagcaacgt
60gatcgtgagt ctaagttgac tggcaagcct attaacatgc aagagcaaat tatcgacggt
120tggttcctag ccagattctg gatcttcaag gatcagaaca ataaccacca gaccaaccgc
180ttcatcagtt ggttcaagga taacttggct agttctaagg gttacgatag tattgctgag
240caaatgggtt tgaagattga ggctttgaac gatatggatg ttaccaacat tgattacact
300tccaagactg gtgatacgat ctacaacggt atttctgagt tgactaacta cactggtact
360actcagaaga tgaagaccga cagcttccag agggattaca ccaagtctga gtcaacctca
420gttaccaacg gacttcagct tggattcaag gttgcagcca agggagttgt ggcacttgct
480ggagcagatt tcgagacctc agttacctac aacctttcat ccaccacaac cgaaacgaac
540accatctccg ataagtttac cgttccatcc caggaagtga cactttcccc aggacacaag
600gccgtggtca agcacgatct caggaagatg gtgtacttcg gaacacacga cctcaaaggc
660gacctcaaag tgggctttaa cgacaaagaa atcgtgcaga agttcatcta tccaaattat
720cgcagcatcg acctcagcga catccgaaag acaatgatcg aaatcgacaa atggaatcac
780gtcaacacaa tcgacttcta tcaactggtc ggcgtaaaga accacatcaa gaacggcgac
840acactctaca tcgacacacc cgccgagttt acattcaatg gggccaatcc ctattatcgg
900gcgacattca cggaatacga cgagaatggg aatccggtac agacgaagat cctgtcgggg
960aattga
96612321PRTArtificial sequencean amino acid sequence translation of SEQ
ID NO 11. 12Met Ile Thr Pro Tyr Ala Glu Ser Tyr Ile Asp Thr Val Gln
Asp Arg 1 5 10 15
Met Lys Gln Arg Asp Arg Glu Ser Lys Leu Thr Gly Lys Pro Ile Asn
20 25 30 Met Gln Glu Gln Ile
Ile Asp Gly Trp Phe Leu Ala Arg Phe Trp Ile 35
40 45 Phe Lys Asp Gln Asn Asn Asn His Gln
Thr Asn Arg Phe Ile Ser Trp 50 55
60 Phe Lys Asp Asn Leu Ala Ser Ser Lys Gly Tyr Asp Ser
Ile Ala Glu 65 70 75
80 Gln Met Gly Leu Lys Ile Glu Ala Leu Asn Asp Met Asp Val Thr Asn
85 90 95 Ile Asp Tyr Thr
Ser Lys Thr Gly Asp Thr Ile Tyr Asn Gly Ile Ser 100
105 110 Glu Leu Thr Asn Tyr Thr Gly Thr Thr
Gln Lys Met Lys Thr Asp Ser 115 120
125 Phe Gln Arg Asp Tyr Thr Lys Ser Glu Ser Thr Ser Val Thr
Asn Gly 130 135 140
Leu Gln Leu Gly Phe Lys Val Ala Ala Lys Gly Val Val Ala Leu Ala 145
150 155 160 Gly Ala Asp Phe Glu
Thr Ser Val Thr Tyr Asn Leu Ser Ser Thr Thr 165
170 175 Thr Glu Thr Asn Thr Ile Ser Asp Lys Phe
Thr Val Pro Ser Gln Glu 180 185
190 Val Thr Leu Ser Pro Gly His Lys Ala Val Val Lys His Asp Leu
Arg 195 200 205 Lys
Met Val Tyr Phe Gly Thr His Asp Leu Lys Gly Asp Leu Lys Val 210
215 220 Gly Phe Asn Asp Lys Glu
Ile Val Gln Lys Phe Ile Tyr Pro Asn Tyr 225 230
235 240 Arg Ser Ile Asp Leu Ser Asp Ile Arg Lys Thr
Met Ile Glu Ile Asp 245 250
255 Lys Trp Asn His Val Asn Thr Ile Asp Phe Tyr Gln Leu Val Gly Val
260 265 270 Lys Asn
His Ile Lys Asn Gly Asp Thr Leu Tyr Ile Asp Thr Pro Ala 275
280 285 Glu Phe Thr Phe Asn Gly Ala
Asn Pro Tyr Tyr Arg Ala Thr Phe Thr 290 295
300 Glu Tyr Asp Glu Asn Gly Asn Pro Val Gln Thr Lys
Ile Leu Ser Gly 305 310 315
320 Asn 13681DNABacillus thuringiensis 13gcaataactc catatgctga
atcttatatt gatactgttc aagatagaat gaaacaaaga 60gatagggaat caaaactaac
tggtaaacca ataaatatgc aagaacaaat aatagatgga 120tggtttttag ctagattctg
gatatttaaa gatcaaaata acaatcatca aacaaataga 180tttatatcct ggtttaaaga
taatcttgct agttcgaagg ggtatgacag tatagcagaa 240caaatgggct taaaaataga
agcattaaat gatatggatg taacaaatat tgattataca 300tctaaaacag gtgataccat
atataatgga atttctgaac taacaaatta tacaggaaca 360acccaaaaaa tgaaaaccga
tagttttcaa agagattata caaaatctga atccacttca 420gtaacaaatg ggttacaatt
aggatttaaa gttgctgcta agggagtagt tgcattagca 480ggtgcagatt ttgaaacaag
tgttacctat aatttatcat ctactacaac tgaaacaaat 540acaatatcgg ataagtttac
tgttccatct caagaagtta cattatcccc aggacataaa 600gcagtggtga aacatgattt
gagaaaaatg gtgtattttg ggactcatga tttaaagggt 660gatttaaaag taggttttaa t
68114227PRTArtificial
sequencean amino acid sequence translation of SEQ ID NO 13. 14Met
Ile Thr Pro Tyr Ala Glu Ser Tyr Ile Asp Thr Val Gln Asp Arg 1
5 10 15 Met Lys Gln Arg Asp Arg
Glu Ser Lys Leu Thr Gly Lys Pro Ile Asn 20
25 30 Met Gln Glu Gln Ile Ile Asp Gly Trp Phe
Leu Ala Arg Phe Trp Ile 35 40
45 Phe Lys Asp Gln Asn Asn Asn His Gln Thr Asn Arg Phe Ile
Ser Trp 50 55 60
Phe Lys Asp Asn Leu Ala Ser Ser Lys Gly Tyr Asp Ser Ile Ala Glu 65
70 75 80 Gln Met Gly Leu Lys
Ile Glu Ala Leu Asn Asp Met Asp Val Thr Asn 85
90 95 Ile Asp Tyr Thr Ser Lys Thr Gly Asp Thr
Ile Tyr Asn Gly Ile Ser 100 105
110 Glu Leu Thr Asn Tyr Thr Gly Thr Thr Gln Lys Met Lys Thr Asp
Ser 115 120 125 Phe
Gln Arg Asp Tyr Thr Lys Ser Glu Ser Thr Ser Val Thr Asn Gly 130
135 140 Leu Gln Leu Gly Phe Lys
Val Ala Ala Lys Gly Val Val Ala Leu Ala 145 150
155 160 Gly Ala Asp Phe Glu Thr Ser Val Thr Tyr Asn
Leu Ser Ser Thr Thr 165 170
175 Thr Glu Thr Asn Thr Ile Ser Asp Lys Phe Thr Val Pro Ser Gln Glu
180 185 190 Val Thr
Leu Ser Pro Gly His Lys Ala Val Val Lys His Asp Leu Arg 195
200 205 Lys Met Val Tyr Phe Gly Thr
His Asp Leu Lys Gly Asp Leu Lys Val 210 215
220 Gly Phe Asn 225 15684DNAArtificial
sequencea recombinant polynucleotide sequence encoding a TIC1501
protein. 15atgattactc cttacgctga gtcttacatt gatactgttc aagatcgcat
gaagcaacgt 60gatcgtgagt ctaagttgac tggcaagcct attaacatgc aagagcaaat
tatcgacggt 120tggttcctag ccagattctg gatcttcaag gatcagaaca ataaccacca
gaccaaccgc 180ttcatcagtt ggttcaagga taacttggct agttctaagg gttacgatag
tattgctgag 240caaatgggtt tgaagattga ggctttgaac gatatggatg ttaccaacat
tgattacact 300tccaagactg gtgatacgat ctacaacggt atttctgagt tgactaacta
cactggtact 360actcagaaga tgaagaccga cagcttccag agggattaca ccaagtctga
gtcaacctca 420gttaccaacg gacttcagct tggattcaag gttgcagcca agggagttgt
ggcacttgct 480ggagcagatt tcgagacctc agttacctac aacctttcat ccaccacaac
cgaaacgaac 540accatctccg ataagtttac cgttccatcc caggaagtga cactttcccc
aggacacaag 600gccgtggtca agcacgatct caggaagatg gtgtacttcg gaacacacga
cctcaaaggc 660gacctcaaag tgggctttaa ctga
68416227PRTArtificial sequencean amino acid sequence
translation of SEQ ID NO 15. 16Met Ile Thr Pro Tyr Ala Glu Ser Tyr
Ile Asp Thr Val Gln Asp Arg 1 5 10
15 Met Lys Gln Arg Asp Arg Glu Ser Lys Leu Thr Gly Lys Pro
Ile Asn 20 25 30
Met Gln Glu Gln Ile Ile Asp Gly Trp Phe Leu Ala Arg Phe Trp Ile
35 40 45 Phe Lys Asp Gln
Asn Asn Asn His Gln Thr Asn Arg Phe Ile Ser Trp 50
55 60 Phe Lys Asp Asn Leu Ala Ser Ser
Lys Gly Tyr Asp Ser Ile Ala Glu 65 70
75 80 Gln Met Gly Leu Lys Ile Glu Ala Leu Asn Asp Met
Asp Val Thr Asn 85 90
95 Ile Asp Tyr Thr Ser Lys Thr Gly Asp Thr Ile Tyr Asn Gly Ile Ser
100 105 110 Glu Leu Thr
Asn Tyr Thr Gly Thr Thr Gln Lys Met Lys Thr Asp Ser 115
120 125 Phe Gln Arg Asp Tyr Thr Lys Ser
Glu Ser Thr Ser Val Thr Asn Gly 130 135
140 Leu Gln Leu Gly Phe Lys Val Ala Ala Lys Gly Val Val
Ala Leu Ala 145 150 155
160 Gly Ala Asp Phe Glu Thr Ser Val Thr Tyr Asn Leu Ser Ser Thr Thr
165 170 175 Thr Glu Thr Asn
Thr Ile Ser Asp Lys Phe Thr Val Pro Ser Gln Glu 180
185 190 Val Thr Leu Ser Pro Gly His Lys Ala
Val Val Lys His Asp Leu Arg 195 200
205 Lys Met Val Tyr Phe Gly Thr His Asp Leu Lys Gly Asp Leu
Lys Val 210 215 220
Gly Phe Asn 225 17903DNABacillus thuringiensis 17gcaataactc
catatgctga atcttatatt gatactgttc aagatagaat gaaacaaaga 60gatagggaat
caaaactaac tggtaaacca ataaatatgc aagaacaaat aatagatgga 120tggtttttag
ctagattctg gatatttaaa gatcaaaata acaatcatca aacaaataga 180tttatatcct
ggtttaaaga taatcttgct agttcgaagg ggtatgacag tatagcagaa 240caaatgggct
taaaaataga agcattaaat gatatggatg taacaaatat tgattataca 300tctaaaacag
gtgataccat atataatgga atttctgaac taacaaatta tacaggaaca 360acccaaaaaa
tgaaaaccga tagttttcaa agagattata caaaatctga atccacttca 420gtaacaaatg
ggttacaatt aggatttaaa gttgctgcta agggagtagt tgcattagca 480ggtgcagatt
ttgaaacaag tgttacctat aatttatcat ctactacaac tgaaacaaat 540acaatatcgg
ataagtttac tgttccatct caagaagtta cattatcccc aggacataaa 600gcagtggtga
aacatgattt gagaaaaatg gtgtattttg ggactcatga tttaaagggt 660gatttaaaag
taggttttaa tgataaagag attgtacaaa aatttattta tccaaattat 720agatcaattg
atttatctga tattcgtaaa acaatgattg aaattgataa atggaatcat 780gtaaatacca
ttgactttta tcaattagtt ggagttaaaa atcatataaa aaatggtgat 840actttatata
tagatacccc ggccgaattt acatttaatg gagctaatcc atattataga 900gca
90318301PRTArtificial sequencean amino acid sequence translation of SEQ
ID NO 17. 18Met Ile Thr Pro Tyr Ala Glu Ser Tyr Ile Asp Thr Val Gln
Asp Arg 1 5 10 15
Met Lys Gln Arg Asp Arg Glu Ser Lys Leu Thr Gly Lys Pro Ile Asn
20 25 30 Met Gln Glu Gln Ile
Ile Asp Gly Trp Phe Leu Ala Arg Phe Trp Ile 35
40 45 Phe Lys Asp Gln Asn Asn Asn His Gln
Thr Asn Arg Phe Ile Ser Trp 50 55
60 Phe Lys Asp Asn Leu Ala Ser Ser Lys Gly Tyr Asp Ser
Ile Ala Glu 65 70 75
80 Gln Met Gly Leu Lys Ile Glu Ala Leu Asn Asp Met Asp Val Thr Asn
85 90 95 Ile Asp Tyr Thr
Ser Lys Thr Gly Asp Thr Ile Tyr Asn Gly Ile Ser 100
105 110 Glu Leu Thr Asn Tyr Thr Gly Thr Thr
Gln Lys Met Lys Thr Asp Ser 115 120
125 Phe Gln Arg Asp Tyr Thr Lys Ser Glu Ser Thr Ser Val Thr
Asn Gly 130 135 140
Leu Gln Leu Gly Phe Lys Val Ala Ala Lys Gly Val Val Ala Leu Ala 145
150 155 160 Gly Ala Asp Phe Glu
Thr Ser Val Thr Tyr Asn Leu Ser Ser Thr Thr 165
170 175 Thr Glu Thr Asn Thr Ile Ser Asp Lys Phe
Thr Val Pro Ser Gln Glu 180 185
190 Val Thr Leu Ser Pro Gly His Lys Ala Val Val Lys His Asp Leu
Arg 195 200 205 Lys
Met Val Tyr Phe Gly Thr His Asp Leu Lys Gly Asp Leu Lys Val 210
215 220 Gly Phe Asn Asp Lys Glu
Ile Val Gln Lys Phe Ile Tyr Pro Asn Tyr 225 230
235 240 Arg Ser Ile Asp Leu Ser Asp Ile Arg Lys Thr
Met Ile Glu Ile Asp 245 250
255 Lys Trp Asn His Val Asn Thr Ile Asp Phe Tyr Gln Leu Val Gly Val
260 265 270 Lys Asn
His Ile Lys Asn Gly Asp Thr Leu Tyr Ile Asp Thr Pro Ala 275
280 285 Glu Phe Thr Phe Asn Gly Ala
Asn Pro Tyr Tyr Arg Ala 290 295 300
19906DNAArtificial sequencea recombinant polynucleotide sequence
encoding a TIC1503 protein. 19atgattactc cttacgctga gtcttacatt
gatactgttc aagatcgcat gaagcaacgt 60gatcgtgagt ctaagttgac tggcaagcct
attaacatgc aagagcaaat tatcgacggt 120tggttcctag ccagattctg gatcttcaag
gatcagaaca ataaccacca gaccaaccgc 180ttcatcagtt ggttcaagga taacttggct
agttctaagg gttacgatag tattgctgag 240caaatgggtt tgaagattga ggctttgaac
gatatggatg ttaccaacat tgattacact 300tccaagactg gtgatacgat ctacaacggt
atttctgagt tgactaacta cactggtact 360actcagaaga tgaagaccga cagcttccag
agggattaca ccaagtctga gtcaacctca 420gttaccaacg gacttcagct tggattcaag
gttgcagcca agggagttgt ggcacttgct 480ggagcagatt tcgagacctc agttacctac
aacctttcat ccaccacaac cgaaacgaac 540accatctccg ataagtttac cgttccatcc
caggaagtga cactttcccc aggacacaag 600gccgtggtca agcacgatct caggaagatg
gtgtacttcg gaacacacga cctcaaaggc 660gacctcaaag tgggctttaa cgacaaagaa
atcgtgcaga agttcatcta tccaaattat 720cgcagcatcg acctcagcga catccgaaag
acaatgatcg aaatcgacaa atggaatcac 780gtcaacacaa tcgacttcta tcaactggtc
ggcgtaaaga accacatcaa gaacggcgac 840acactctaca tcgacacacc cgccgagttt
acattcaatg gggccaatcc ctattatcgg 900gcgtga
90620301PRTArtificial sequencean amino
acid sequence translation of SEQ ID NO 19. 20Met Ile Thr Pro Tyr Ala
Glu Ser Tyr Ile Asp Thr Val Gln Asp Arg 1 5
10 15 Met Lys Gln Arg Asp Arg Glu Ser Lys Leu Thr
Gly Lys Pro Ile Asn 20 25
30 Met Gln Glu Gln Ile Ile Asp Gly Trp Phe Leu Ala Arg Phe Trp
Ile 35 40 45 Phe
Lys Asp Gln Asn Asn Asn His Gln Thr Asn Arg Phe Ile Ser Trp 50
55 60 Phe Lys Asp Asn Leu Ala
Ser Ser Lys Gly Tyr Asp Ser Ile Ala Glu 65 70
75 80 Gln Met Gly Leu Lys Ile Glu Ala Leu Asn Asp
Met Asp Val Thr Asn 85 90
95 Ile Asp Tyr Thr Ser Lys Thr Gly Asp Thr Ile Tyr Asn Gly Ile Ser
100 105 110 Glu Leu
Thr Asn Tyr Thr Gly Thr Thr Gln Lys Met Lys Thr Asp Ser 115
120 125 Phe Gln Arg Asp Tyr Thr Lys
Ser Glu Ser Thr Ser Val Thr Asn Gly 130 135
140 Leu Gln Leu Gly Phe Lys Val Ala Ala Lys Gly Val
Val Ala Leu Ala 145 150 155
160 Gly Ala Asp Phe Glu Thr Ser Val Thr Tyr Asn Leu Ser Ser Thr Thr
165 170 175 Thr Glu Thr
Asn Thr Ile Ser Asp Lys Phe Thr Val Pro Ser Gln Glu 180
185 190 Val Thr Leu Ser Pro Gly His Lys
Ala Val Val Lys His Asp Leu Arg 195 200
205 Lys Met Val Tyr Phe Gly Thr His Asp Leu Lys Gly Asp
Leu Lys Val 210 215 220
Gly Phe Asn Asp Lys Glu Ile Val Gln Lys Phe Ile Tyr Pro Asn Tyr 225
230 235 240 Arg Ser Ile Asp
Leu Ser Asp Ile Arg Lys Thr Met Ile Glu Ile Asp 245
250 255 Lys Trp Asn His Val Asn Thr Ile Asp
Phe Tyr Gln Leu Val Gly Val 260 265
270 Lys Asn His Ile Lys Asn Gly Asp Thr Leu Tyr Ile Asp Thr
Pro Ala 275 280 285
Glu Phe Thr Phe Asn Gly Ala Asn Pro Tyr Tyr Arg Ala 290
295 300 21774DNABacillus thuringiensis 21atggacattc
aagatgtaat agaattaatt agtgactatg ccaacaagtg gaaggaacaa 60caaaatggaa
gtcgtagtga catgaaattc attgatgcac gctttgcgaa caatgcatta 120aaagtagaag
ctgatcgtac tatgtacctg gagcctacag agcaagatat gccagatgta 180aaattagata
cccaagtgtt taccaatact tccacacagc ccaaaactgt ccagtttgat 240atgtttggag
aatacgatac ttggcaaaca tggagaatcg aagatggtgc aacagaagca 300ggaaaatgtc
gctttgccat tgagccattg tttagatcag aggatgtttc tagtccacta 360tctttatttt
taaacgacac acttaccctc tcagggaaaa agccattttc cgttgagggt 420cgtgatttta
cagttcgacc acgtttcaaa gtaacgggta cactcatggc aaaacccaaa 480acatcaaccc
gtgcttttga cgtaaaacgt gacgtcacag gatatgtggg acttgtcaca 540actttagcat
ccggagaact gcaagaatcg ttacacaatg tgggcgctat tttccaacaa 600tattatagtc
cctatatcga agtaaatggt ggaagagtga catttcatga tcgtggtgaa 660tatcaatcat
tagatgtaag cagcatctat attcatattt ttgcagagag cttagatatt 720cccggattaa
cggaggaata taatatttat gacctaagca atggacgggt ataa
77422257PRTBacillus thuringiensis 22Met Asp Ile Gln Asp Val Ile Glu Leu
Ile Ser Asp Tyr Ala Asn Lys 1 5 10
15 Trp Lys Glu Gln Gln Asn Gly Ser Arg Ser Asp Met Lys Phe
Ile Asp 20 25 30
Ala Arg Phe Ala Asn Asn Ala Leu Lys Val Glu Ala Asp Arg Thr Met
35 40 45 Tyr Leu Glu Pro
Thr Glu Gln Asp Met Pro Asp Val Lys Leu Asp Thr 50
55 60 Gln Val Phe Thr Asn Thr Ser Thr
Gln Pro Lys Thr Val Gln Phe Asp 65 70
75 80 Met Phe Gly Glu Tyr Asp Thr Trp Gln Thr Trp Arg
Ile Glu Asp Gly 85 90
95 Ala Thr Glu Ala Gly Lys Cys Arg Phe Ala Ile Glu Pro Leu Phe Arg
100 105 110 Ser Glu Asp
Val Ser Ser Pro Leu Ser Leu Phe Leu Asn Asp Thr Leu 115
120 125 Thr Leu Ser Gly Lys Lys Pro Phe
Ser Val Glu Gly Arg Asp Phe Thr 130 135
140 Val Arg Pro Arg Phe Lys Val Thr Gly Thr Leu Met Ala
Lys Pro Lys 145 150 155
160 Thr Ser Thr Arg Ala Phe Asp Val Lys Arg Asp Val Thr Gly Tyr Val
165 170 175 Gly Leu Val Thr
Thr Leu Ala Ser Gly Glu Leu Gln Glu Ser Leu His 180
185 190 Asn Val Gly Ala Ile Phe Gln Gln Tyr
Tyr Ser Pro Tyr Ile Glu Val 195 200
205 Asn Gly Gly Arg Val Thr Phe His Asp Arg Gly Glu Tyr Gln
Ser Leu 210 215 220
Asp Val Ser Ser Ile Tyr Ile His Ile Phe Ala Glu Ser Leu Asp Ile 225
230 235 240 Pro Gly Leu Thr Glu
Glu Tyr Asn Ile Tyr Asp Leu Ser Asn Gly Arg 245
250 255 Val 23774DNAArtificial
sequenceSynthetic 23atggacattc aagatgtgat tgagttgatt tctgattacg
ccaacaagtg gaaggagcaa 60cagaacggta gtcgtagtga catgaagttc attgatgctc
gcttcgccaa caacgctttg 120aaggttgagg ctgatcgcac tatgtaccta gagcctactg
agcaagacat gcctgatgtt 180aagttggata ctcaagtgtt cacgaacacc agtacccaac
ctaagactgt tcagttcgac 240atgttcggtg agtacgatac ctggcagacc tggagaattg
aggatggtgc taccgaggcg 300ggcaagtgca gattcgcaat tgagccattg ttccgctctg
aggatgtttc ttcaccactt 360tctctcttcc ttaatgacac ccttaccctt tctggtaaga
aaccattcag cgttgaagga 420agggacttta ccgtgaggcc aaggtttaaa gtgaccggaa
cactcatggc caaacccaag 480acttcaaccc gtgcctttga cgtgaaacgc gacgtgacag
gatacgtggg actcgtgaca 540acgctcgcct caggagaact ccaggaatca ctgcacaatg
tcggtgccat ctttcagcag 600tactacagcc cctacatcga agtcaatggc gggcgcgtca
cattccatga ccgaggcgag 660tatcagtccc tagacgtatc cagcatctac atccacatct
ttgcggaaag cctcgacata 720cctgggctca cggaggagta caacatctac gacctgtcga
atgggcgggt atga 77424257PRTArtificial sequenceSynthetic 24Met
Asp Ile Gln Asp Val Ile Glu Leu Ile Ser Asp Tyr Ala Asn Lys 1
5 10 15 Trp Lys Glu Gln Gln Asn
Gly Ser Arg Ser Asp Met Lys Phe Ile Asp 20
25 30 Ala Arg Phe Ala Asn Asn Ala Leu Lys Val
Glu Ala Asp Arg Thr Met 35 40
45 Tyr Leu Glu Pro Thr Glu Gln Asp Met Pro Asp Val Lys Leu
Asp Thr 50 55 60
Gln Val Phe Thr Asn Thr Ser Thr Gln Pro Lys Thr Val Gln Phe Asp 65
70 75 80 Met Phe Gly Glu Tyr
Asp Thr Trp Gln Thr Trp Arg Ile Glu Asp Gly 85
90 95 Ala Thr Glu Ala Gly Lys Cys Arg Phe Ala
Ile Glu Pro Leu Phe Arg 100 105
110 Ser Glu Asp Val Ser Ser Pro Leu Ser Leu Phe Leu Asn Asp Thr
Leu 115 120 125 Thr
Leu Ser Gly Lys Lys Pro Phe Ser Val Glu Gly Arg Asp Phe Thr 130
135 140 Val Arg Pro Arg Phe Lys
Val Thr Gly Thr Leu Met Ala Lys Pro Lys 145 150
155 160 Thr Ser Thr Arg Ala Phe Asp Val Lys Arg Asp
Val Thr Gly Tyr Val 165 170
175 Gly Leu Val Thr Thr Leu Ala Ser Gly Glu Leu Gln Glu Ser Leu His
180 185 190 Asn Val
Gly Ala Ile Phe Gln Gln Tyr Tyr Ser Pro Tyr Ile Glu Val 195
200 205 Asn Gly Gly Arg Val Thr Phe
His Asp Arg Gly Glu Tyr Gln Ser Leu 210 215
220 Asp Val Ser Ser Ile Tyr Ile His Ile Phe Ala Glu
Ser Leu Asp Ile 225 230 235
240 Pro Gly Leu Thr Glu Glu Tyr Asn Ile Tyr Asp Leu Ser Asn Gly Arg
245 250 255 Val
25900DNABacillus thuringiensis 25atggctattt ttaattttga cgcaaaagta
gtagagttta ttaattggtg gacagcagag 60ttcggtggca aagatcccag aaatattcaa
attggttatg agaatcgaga catcaatgtt 120gttccatcat caagtaaccc tagtgtcaat
gtaataccta agttagcaag atcatctgtt 180caagaattgc aaaataatac aagtgtaacg
cagacacaag agctggcatt ttcagaaact 240acaactgaaa gtcagtcttc tacaacaaca
catggggctt cattttctac aacggtcacc 300tctgttacgc aatttacagc tgaagttaat
ttcaaagcga ttggttcctc aattgagcaa 360actatcggcg tttctatgac aggagattat
aattacagtt cttcactaac aaaaacaaca 420gaaaagagta gatcttggac tctcacacaa
ccggtagttg tcccaccttt ttcacgtgta 480acttgcacat tattgatata taatgcacca
ttttcagtac ctgtggactt aaattgtaat 540gtattcggta cacttggcgg agatttttta
gctagctaca cttacactgt tattagtact 600ggtcgaacag taaatactag tataactgct
agtcaaatga ctcttacctc ttggccaggt 660aaaccatctg aaattattgg tatcgcacca
aagcatggac ttatttttaa agggacaggt 720acacaggctg cagtacatgg attatactca
accgttaaat ttgttgaatc cccattgcca 780ggtcaccaag gagaaaaaag aacgtattac
cttccagcgc aacctgtaaa tgaagatgat 840ctgatttctt ctgtatttag taacattcca
attattaatc ctgtttctaa tctataatga 90026298PRTBacillus thuringiensis
26Met Ala Ile Phe Asn Phe Asp Ala Lys Val Val Glu Phe Ile Asn Trp 1
5 10 15 Trp Thr Ala Glu
Phe Gly Gly Lys Asp Pro Arg Asn Ile Gln Ile Gly 20
25 30 Tyr Glu Asn Arg Asp Ile Asn Val Val
Pro Ser Ser Ser Asn Pro Ser 35 40
45 Val Asn Val Ile Pro Lys Leu Ala Arg Ser Ser Val Gln Glu
Leu Gln 50 55 60
Asn Asn Thr Ser Val Thr Gln Thr Gln Glu Leu Ala Phe Ser Glu Thr 65
70 75 80 Thr Thr Glu Ser Gln
Ser Ser Thr Thr Thr His Gly Ala Ser Phe Ser 85
90 95 Thr Thr Val Thr Ser Val Thr Gln Phe Thr
Ala Glu Val Asn Phe Lys 100 105
110 Ala Ile Gly Ser Ser Ile Glu Gln Thr Ile Gly Val Ser Met Thr
Gly 115 120 125 Asp
Tyr Asn Tyr Ser Ser Ser Leu Thr Lys Thr Thr Glu Lys Ser Arg 130
135 140 Ser Trp Thr Leu Thr Gln
Pro Val Val Val Pro Pro Phe Ser Arg Val 145 150
155 160 Thr Cys Thr Leu Leu Ile Tyr Asn Ala Pro Phe
Ser Val Pro Val Asp 165 170
175 Leu Asn Cys Asn Val Phe Gly Thr Leu Gly Gly Asp Phe Leu Ala Ser
180 185 190 Tyr Thr
Tyr Thr Val Ile Ser Thr Gly Arg Thr Val Asn Thr Ser Ile 195
200 205 Thr Ala Ser Gln Met Thr Leu
Thr Ser Trp Pro Gly Lys Pro Ser Glu 210 215
220 Ile Ile Gly Ile Ala Pro Lys His Gly Leu Ile Phe
Lys Gly Thr Gly 225 230 235
240 Thr Gln Ala Ala Val His Gly Leu Tyr Ser Thr Val Lys Phe Val Glu
245 250 255 Ser Pro Leu
Pro Gly His Gln Gly Glu Lys Arg Thr Tyr Tyr Leu Pro 260
265 270 Ala Gln Pro Val Asn Glu Asp Asp
Leu Ile Ser Ser Val Phe Ser Asn 275 280
285 Ile Pro Ile Ile Asn Pro Val Ser Asn Leu 290
295 27897DNAArtificial sequenceSynthetic
27atggctatct tcaacttcga cgccaaggtg gtcgagttca tcaactggtg gactgctgag
60ttcggcggca aagatccaag gaacatccag atcggctacg agaatcgtga catcaacgtg
120gtccctagct ctagcaaccc tagcgtgaat gtgatcccga agctcgctag gtccagcgta
180caggagcttc agaacaacac cagcgttacc cagactcagg agctggcctt ctccgaaacg
240acaaccgaat cccagtcctc tactaccact cacggcgctt cctttagcac taccgtgacc
300tcagtgaccc aattcaccgc cgaggtcaac ttcaaggcaa tcggtagctc tattgagcaa
360acaatcggcg tgagcatgac aggcgattac aactactcct cttcgttgac caagacaacc
420gagaaatcac gctcctggac gctgacgcag cctgttgtgg ttcctccctt ctctcgcgtc
480acgtgcaccc tcctaatcta caacgcgccc ttcagtgttc cagtcgatct caattgcaac
540gtcttcggaa cgcttggagg cgactttctg gcctcgtaca cttacaccgt gatctcaacg
600ggaagaactg tcaacacctc gattaccgcc tctcagatga ctctcaccag ttggcctggg
660aagcccagtg agatcattgg tatcgctccg aagcacggcc tcatcttcaa gggcactggt
720acacaggcgg ccgtccacgg gttgtactca acggtcaagt tcgttgaatc gccgctccct
780ggccatcagg gcgagaaacg gacctactat ctgccagcac aaccagtgaa cgaggacgat
840cttatttcct cggtgttctc caacattccg atcatcaacc cagtgtccaa tctgtga
89728298PRTArtificial sequenceSynthetic 28Met Ala Ile Phe Asn Phe Asp Ala
Lys Val Val Glu Phe Ile Asn Trp 1 5 10
15 Trp Thr Ala Glu Phe Gly Gly Lys Asp Pro Arg Asn Ile
Gln Ile Gly 20 25 30
Tyr Glu Asn Arg Asp Ile Asn Val Val Pro Ser Ser Ser Asn Pro Ser
35 40 45 Val Asn Val Ile
Pro Lys Leu Ala Arg Ser Ser Val Gln Glu Leu Gln 50
55 60 Asn Asn Thr Ser Val Thr Gln Thr
Gln Glu Leu Ala Phe Ser Glu Thr 65 70
75 80 Thr Thr Glu Ser Gln Ser Ser Thr Thr Thr His Gly
Ala Ser Phe Ser 85 90
95 Thr Thr Val Thr Ser Val Thr Gln Phe Thr Ala Glu Val Asn Phe Lys
100 105 110 Ala Ile Gly
Ser Ser Ile Glu Gln Thr Ile Gly Val Ser Met Thr Gly 115
120 125 Asp Tyr Asn Tyr Ser Ser Ser Leu
Thr Lys Thr Thr Glu Lys Ser Arg 130 135
140 Ser Trp Thr Leu Thr Gln Pro Val Val Val Pro Pro Phe
Ser Arg Val 145 150 155
160 Thr Cys Thr Leu Leu Ile Tyr Asn Ala Pro Phe Ser Val Pro Val Asp
165 170 175 Leu Asn Cys Asn
Val Phe Gly Thr Leu Gly Gly Asp Phe Leu Ala Ser 180
185 190 Tyr Thr Tyr Thr Val Ile Ser Thr Gly
Arg Thr Val Asn Thr Ser Ile 195 200
205 Thr Ala Ser Gln Met Thr Leu Thr Ser Trp Pro Gly Lys Pro
Ser Glu 210 215 220
Ile Ile Gly Ile Ala Pro Lys His Gly Leu Ile Phe Lys Gly Thr Gly 225
230 235 240 Thr Gln Ala Ala Val
His Gly Leu Tyr Ser Thr Val Lys Phe Val Glu 245
250 255 Ser Pro Leu Pro Gly His Gln Gly Glu Lys
Arg Thr Tyr Tyr Leu Pro 260 265
270 Ala Gln Pro Val Asn Glu Asp Asp Leu Ile Ser Ser Val Phe Ser
Asn 275 280 285 Ile
Pro Ile Ile Asn Pro Val Ser Asn Leu 290 295
29807DNABacillus thuringiensis 29atgggaatta tcaacattca agacgaaatt
aatgactaca tgaaaggtat gtatggtgca 60acatctgtta aaagcacata tgacccctca
ttcaaagtat ttaacgaatc tgtaacacct 120caatatgatg tgatttcaac agaacctgta
aataatcata ttactactaa agcaataaat 180aatccagcgt cttcagaagt aaccagtaca
gtaaccttca catggacgga aaccgacact 240gtaacctctg cagtgactaa agggtataaa
gtcggtggtt cagtaagctc aaaagcaact 300tttaaatttg cttttgttac ttctgatgtt
actgtaactg tatcagcaga atataattat 360agtacaacag atacaacaac aaaaacagat
acacgcacat ggacggattc gacgacagta 420aaagcccctc caagaactaa tgtagaagtt
gcatatatta tccaaactgg aaattataac 480gttccggtta atgtagagtc tgatatgact
ggaacgctat tttgcagagg gtatagagat 540ggtgcactaa ttgcagcgac ttatatttct
ataacagatt tagcagatta caagcctaat 600ttgggtctta caaataaagg ggatggggtt
gctcatttta aaggtgaagg ttatatagag 660ggtgcacaag gcttaagaag ctatattcaa
gttacagaat atccaatgga tgataaagac 720agacgttcga caccaaaaac ttatacaatt
gaaggttcat tagcacccaa tgttacttta 780ataaatgata gaaaggaagg tagataa
80730268PRTBacillus thuringiensis 30Met
Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asp Tyr Met Lys Gly 1
5 10 15 Met Tyr Gly Ala Thr Ser
Val Lys Ser Thr Tyr Asp Pro Ser Phe Lys 20
25 30 Val Phe Asn Glu Ser Val Thr Pro Gln Tyr
Asp Val Ile Ser Thr Glu 35 40
45 Pro Val Asn Asn His Ile Thr Thr Lys Ala Ile Asn Asn Pro
Ala Ser 50 55 60
Ser Glu Val Thr Ser Thr Val Thr Phe Thr Trp Thr Glu Thr Asp Thr 65
70 75 80 Val Thr Ser Ala Val
Thr Lys Gly Tyr Lys Val Gly Gly Ser Val Ser 85
90 95 Ser Lys Ala Thr Phe Lys Phe Ala Phe Val
Thr Ser Asp Val Thr Val 100 105
110 Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Asp Thr Thr Thr
Lys 115 120 125 Thr
Asp Thr Arg Thr Trp Thr Asp Ser Thr Thr Val Lys Ala Pro Pro 130
135 140 Arg Thr Asn Val Glu Val
Ala Tyr Ile Ile Gln Thr Gly Asn Tyr Asn 145 150
155 160 Val Pro Val Asn Val Glu Ser Asp Met Thr Gly
Thr Leu Phe Cys Arg 165 170
175 Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Thr Tyr Ile Ser Ile Thr
180 185 190 Asp Leu
Ala Asp Tyr Lys Pro Asn Leu Gly Leu Thr Asn Lys Gly Asp 195
200 205 Gly Val Ala His Phe Lys Gly
Glu Gly Tyr Ile Glu Gly Ala Gln Gly 210 215
220 Leu Arg Ser Tyr Ile Gln Val Thr Glu Tyr Pro Met
Asp Asp Lys Asp 225 230 235
240 Arg Arg Ser Thr Pro Lys Thr Tyr Thr Ile Glu Gly Ser Leu Ala Pro
245 250 255 Asn Val Thr
Leu Ile Asn Asp Arg Lys Glu Gly Arg 260 265
31807DNAArtificial sequenceSynthetic 31atgggcatta tcaacattca
agatgagatt aacgattaca tgaagggtat gtacggtgct 60accagtgtga agtccactta
cgatcctagt ttcaaggtct tcaacgagtc tgtgactcct 120caatacgatg tgatttctac
tgagcctgtg aacaatcaca ttactactaa ggctattaac 180aatcctgcta gttctgaggt
tactagcact gttactttca cttggactga gactgatact 240gttactagcg ctgttactaa
gggttacaag gttggtggat ctgtttcttc aaaggctacc 300ttcaagtttg ctttcgttac
ctcagatgtt accgttaccg tgagcgctga gtacaactac 360tcaaccacag ataccaccac
caagaccgat accagaacct ggaccgattc aaccacagtg 420aaggcaccac ctcgcaccaa
cgtggaggtc gcctacatta tccaaacagg caactacaat 480gtcccagtca atgtcgaatc
cgacatgaca ggaacactct tctgccgtgg ctacagggat 540ggagcactta ttgcagccac
ctacatctcc atcacagacc ttgccgacta caagccaaat 600ctcggactca caaacaaggg
agacggcgtc gcccacttca aaggcgaagg ctacatcgaa 660ggtgcccagg gtctaaggag
ctacatccaa gtgacagaat acccaatgga cgacaaagac 720cgtcgctcca cacccaagac
gtacaccatc gaagggtcgt tagcgccgaa tgtaacgctg 780ataaatgacc gaaaggaagg
gcggtga 80732268PRTArtificial
sequenceSynthetic 32Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asp Tyr
Met Lys Gly 1 5 10 15
Met Tyr Gly Ala Thr Ser Val Lys Ser Thr Tyr Asp Pro Ser Phe Lys
20 25 30 Val Phe Asn Glu
Ser Val Thr Pro Gln Tyr Asp Val Ile Ser Thr Glu 35
40 45 Pro Val Asn Asn His Ile Thr Thr Lys
Ala Ile Asn Asn Pro Ala Ser 50 55
60 Ser Glu Val Thr Ser Thr Val Thr Phe Thr Trp Thr Glu
Thr Asp Thr 65 70 75
80 Val Thr Ser Ala Val Thr Lys Gly Tyr Lys Val Gly Gly Ser Val Ser
85 90 95 Ser Lys Ala Thr
Phe Lys Phe Ala Phe Val Thr Ser Asp Val Thr Val 100
105 110 Thr Val Ser Ala Glu Tyr Asn Tyr Ser
Thr Thr Asp Thr Thr Thr Lys 115 120
125 Thr Asp Thr Arg Thr Trp Thr Asp Ser Thr Thr Val Lys Ala
Pro Pro 130 135 140
Arg Thr Asn Val Glu Val Ala Tyr Ile Ile Gln Thr Gly Asn Tyr Asn 145
150 155 160 Val Pro Val Asn Val
Glu Ser Asp Met Thr Gly Thr Leu Phe Cys Arg 165
170 175 Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala
Thr Tyr Ile Ser Ile Thr 180 185
190 Asp Leu Ala Asp Tyr Lys Pro Asn Leu Gly Leu Thr Asn Lys Gly
Asp 195 200 205 Gly
Val Ala His Phe Lys Gly Glu Gly Tyr Ile Glu Gly Ala Gln Gly 210
215 220 Leu Arg Ser Tyr Ile Gln
Val Thr Glu Tyr Pro Met Asp Asp Lys Asp 225 230
235 240 Arg Arg Ser Thr Pro Lys Thr Tyr Thr Ile Glu
Gly Ser Leu Ala Pro 245 250
255 Asn Val Thr Leu Ile Asn Asp Arg Lys Glu Gly Arg 260
265 33381DNABacillus thuringiensis
33atgacagtat ataacgtaac ttttaccatt aaattcttta atcacggtga atgggggggg
60ccagaacctt acggtaagat atatgcatat cttcaaaatc cagatcataa tttcgaaatt
120tggtcacaag ataattgggg gaaggatacg cctgagaaaa gttctcacac tcaaacaatt
180aaaataagta gcccaacagg ggggcctata gaccaaatgt gtttttatgg tgatgtaaaa
240gaattcgacg taggaaattc agatgatgtt ctcgcctctc caagtcaaaa agtatgcagt
300acgcctggca caacaataag gcttaacgga gatgagagtg gttcttatat agagattagt
360tattccttgg ccccagctta a
38134126PRTBacillus thuringiensis 34Met Thr Val Tyr Asn Val Thr Phe Thr
Ile Lys Phe Phe Asn His Gly 1 5 10
15 Glu Trp Gly Gly Pro Glu Pro Tyr Gly Lys Ile Tyr Ala Tyr
Leu Gln 20 25 30
Asn Pro Asp His Asn Phe Glu Ile Trp Ser Gln Asp Asn Trp Gly Lys
35 40 45 Asp Thr Pro Glu
Lys Ser Ser His Thr Gln Thr Ile Lys Ile Ser Ser 50
55 60 Pro Thr Gly Gly Pro Ile Asp Gln
Met Cys Phe Tyr Gly Asp Val Lys 65 70
75 80 Glu Phe Asp Val Gly Asn Ser Asp Asp Val Leu Ala
Ser Pro Ser Gln 85 90
95 Lys Val Cys Ser Thr Pro Gly Thr Thr Ile Arg Leu Asn Gly Asp Glu
100 105 110 Ser Gly Ser
Tyr Ile Glu Ile Ser Tyr Ser Leu Ala Pro Ala 115
120 125 35381DNAArtificial sequenceSynthetic
35atgaccgtgt acaacgtgac cttcaccatt aagttcttca atcacggtga gtggggcggt
60cctgagccct acggcaaaat ctacgcctat ctccagaacc ctgaccacaa cttcgaaatc
120tggtcccagg acaactgggg caaggacact cccgagaagt ccagccatac gcagaccatc
180aagatcagca gcccgactgg cggcccgatt gaccagatgt gcttctacgg cgacgtcaag
240gaatttgacg tgggaaactc agatgacgtc ctcgccagcc cgtcccagaa ggtttgctcg
300acgccaggaa ctaccatccg cctgaatggc gatgaatctg ggtcgtacat cgagatcagt
360tacagccttg cgcctgcatg a
38136126PRTArtificial sequenceSynthetic 36Met Thr Val Tyr Asn Val Thr Phe
Thr Ile Lys Phe Phe Asn His Gly 1 5 10
15 Glu Trp Gly Gly Pro Glu Pro Tyr Gly Lys Ile Tyr Ala
Tyr Leu Gln 20 25 30
Asn Pro Asp His Asn Phe Glu Ile Trp Ser Gln Asp Asn Trp Gly Lys
35 40 45 Asp Thr Pro Glu
Lys Ser Ser His Thr Gln Thr Ile Lys Ile Ser Ser 50
55 60 Pro Thr Gly Gly Pro Ile Asp Gln
Met Cys Phe Tyr Gly Asp Val Lys 65 70
75 80 Glu Phe Asp Val Gly Asn Ser Asp Asp Val Leu Ala
Ser Pro Ser Gln 85 90
95 Lys Val Cys Ser Thr Pro Gly Thr Thr Ile Arg Leu Asn Gly Asp Glu
100 105 110 Ser Gly Ser
Tyr Ile Glu Ile Ser Tyr Ser Leu Ala Pro Ala 115
120 125 37906DNABacillus thuringiensis 37atgaacaata
attctaaatt tttattctca cctataaatg ctaatggaaa taatccttat 60tttttagatg
tagataattg gaaagatgta ttttgttatc ttgtaaatac tctggaaatg 120aaatatacaa
aagtagttaa taaatctcaa ttaatagatg cattatttcc agatcctaat 180gatcaaagaa
taggtccatt ttattggaat atatatgata attataaatt agataatgta 240ttaaaaccat
tatatgattt tcatagttca ttattaggaa atattcttga atggggatta 300tttggaacaa
taagaatggg tagtttggct tatcatattc aagattttgt aaatatcata 360tgtgtaaaac
atataaatga atttaatcaa attatagatg atttacaaca agataatgtt 420tcaacaaaaa
ctgttgataa atttagaaat atatgtattg aactacgtac taaatcactt 480ctttttcctc
aagaagtaaa taaaatatta gatgcaagta gaaatcttgc aaataatctt 540ataaaagtta
atgctatttt agattcaaca gtgcaacaat ttgaaaacgt ttatcaatca 600tcacaatcaa
ataacgataa atattatttg agaatcgtaa aagaatattt aggaaacgaa 660caacaagatt
tagaaaaaca aactcctaat catcttaata ctcttcaaaa attacgtgga 720atatggactg
tttttggaga taatttagaa aaattaattc atgcttgtga tgatgatctt 780atagattttg
atgcaatgat agctagtata aatttagagg atgctataac ttcttggaaa 840gaagtaaaaa
ataatataga taaatttata ccagaatggg aaatctttaa aaaacaagga 900tgctaa
90638301PRTBacillus thuringiensis 38Met Asn Asn Asn Ser Lys Phe Leu Phe
Ser Pro Ile Asn Ala Asn Gly 1 5 10
15 Asn Asn Pro Tyr Phe Leu Asp Val Asp Asn Trp Lys Asp Val
Phe Cys 20 25 30
Tyr Leu Val Asn Thr Leu Glu Met Lys Tyr Thr Lys Val Val Asn Lys
35 40 45 Ser Gln Leu Ile
Asp Ala Leu Phe Pro Asp Pro Asn Asp Gln Arg Ile 50
55 60 Gly Pro Phe Tyr Trp Asn Ile Tyr
Asp Asn Tyr Lys Leu Asp Asn Val 65 70
75 80 Leu Lys Pro Leu Tyr Asp Phe His Ser Ser Leu Leu
Gly Asn Ile Leu 85 90
95 Glu Trp Gly Leu Phe Gly Thr Ile Arg Met Gly Ser Leu Ala Tyr His
100 105 110 Ile Gln Asp
Phe Val Asn Ile Ile Cys Val Lys His Ile Asn Glu Phe 115
120 125 Asn Gln Ile Ile Asp Asp Leu Gln
Gln Asp Asn Val Ser Thr Lys Thr 130 135
140 Val Asp Lys Phe Arg Asn Ile Cys Ile Glu Leu Arg Thr
Lys Ser Leu 145 150 155
160 Leu Phe Pro Gln Glu Val Asn Lys Ile Leu Asp Ala Ser Arg Asn Leu
165 170 175 Ala Asn Asn Leu
Ile Lys Val Asn Ala Ile Leu Asp Ser Thr Val Gln 180
185 190 Gln Phe Glu Asn Val Tyr Gln Ser Ser
Gln Ser Asn Asn Asp Lys Tyr 195 200
205 Tyr Leu Arg Ile Val Lys Glu Tyr Leu Gly Asn Glu Gln Gln
Asp Leu 210 215 220
Glu Lys Gln Thr Pro Asn His Leu Asn Thr Leu Gln Lys Leu Arg Gly 225
230 235 240 Ile Trp Thr Val Phe
Gly Asp Asn Leu Glu Lys Leu Ile His Ala Cys 245
250 255 Asp Asp Asp Leu Ile Asp Phe Asp Ala Met
Ile Ala Ser Ile Asn Leu 260 265
270 Glu Asp Ala Ile Thr Ser Trp Lys Glu Val Lys Asn Asn Ile Asp
Lys 275 280 285 Phe
Ile Pro Glu Trp Glu Ile Phe Lys Lys Gln Gly Cys 290
295 300 39906DNAArtificial sequenceSynthetic
39atgaacaaca actctaagtt cctcttctcg cccatcaacg ctaacggcaa caacccttac
60ttcctagacg tggacaactg gaaggacgtc ttctgctacc tagtgaacac ccttgagatg
120aagtacacta aggtggtgaa caagtcccag ctcattgacg ccctcttccc agatcctaac
180gatcagcgta tcggaccctt ctactggaac atctacgata actacaagct cgacaacgtc
240ctcaagccac tgtacgactt tcacagctca ctcctgggca acatccttga gtggggactc
300tttgggacca ttcgcatggg ttctctggcc taccacatcc aagatttcgt caacattatc
360tgcgttaagc acatcaacga gttcaaccag ataattgatg acctccagca agataacgtc
420agcactaaga cggtggacaa gttccgcaac atctgcatcg aactccgcac gaagtccctt
480ctgttccctc aggaggtcaa taagattctg gatgcgtcac ggaacctcgc aaataacctc
540atcaaggtta atgcaatcct cgatagtacc gtccaacagt tcgagaacgt ttaccagtcg
600tctcagtcta ataacgacaa gtactatttg agaatcgtaa aggagtatct cgggaatgag
660cagcaagacc tggagaagca gacaccgaat cacctgaaca cccttcagaa gctccgtggc
720atctggacag tcttcggcga caatcttgag aagctcattc atgcttgtga tgacgatctc
780atcgacttcg acgctatgat cgcctccatc aacttggaag atgcgatcac ttcgtggaag
840gaggttaaga ataacattga caaattcatc cctgaatggg aaatcttcaa gaaacagggc
900tgctga
90640301PRTArtificial sequenceSynthetic 40Met Asn Asn Asn Ser Lys Phe Leu
Phe Ser Pro Ile Asn Ala Asn Gly 1 5 10
15 Asn Asn Pro Tyr Phe Leu Asp Val Asp Asn Trp Lys Asp
Val Phe Cys 20 25 30
Tyr Leu Val Asn Thr Leu Glu Met Lys Tyr Thr Lys Val Val Asn Lys
35 40 45 Ser Gln Leu Ile
Asp Ala Leu Phe Pro Asp Pro Asn Asp Gln Arg Ile 50
55 60 Gly Pro Phe Tyr Trp Asn Ile Tyr
Asp Asn Tyr Lys Leu Asp Asn Val 65 70
75 80 Leu Lys Pro Leu Tyr Asp Phe His Ser Ser Leu Leu
Gly Asn Ile Leu 85 90
95 Glu Trp Gly Leu Phe Gly Thr Ile Arg Met Gly Ser Leu Ala Tyr His
100 105 110 Ile Gln Asp
Phe Val Asn Ile Ile Cys Val Lys His Ile Asn Glu Phe 115
120 125 Asn Gln Ile Ile Asp Asp Leu Gln
Gln Asp Asn Val Ser Thr Lys Thr 130 135
140 Val Asp Lys Phe Arg Asn Ile Cys Ile Glu Leu Arg Thr
Lys Ser Leu 145 150 155
160 Leu Phe Pro Gln Glu Val Asn Lys Ile Leu Asp Ala Ser Arg Asn Leu
165 170 175 Ala Asn Asn Leu
Ile Lys Val Asn Ala Ile Leu Asp Ser Thr Val Gln 180
185 190 Gln Phe Glu Asn Val Tyr Gln Ser Ser
Gln Ser Asn Asn Asp Lys Tyr 195 200
205 Tyr Leu Arg Ile Val Lys Glu Tyr Leu Gly Asn Glu Gln Gln
Asp Leu 210 215 220
Glu Lys Gln Thr Pro Asn His Leu Asn Thr Leu Gln Lys Leu Arg Gly 225
230 235 240 Ile Trp Thr Val Phe
Gly Asp Asn Leu Glu Lys Leu Ile His Ala Cys 245
250 255 Asp Asp Asp Leu Ile Asp Phe Asp Ala Met
Ile Ala Ser Ile Asn Leu 260 265
270 Glu Asp Ala Ile Thr Ser Trp Lys Glu Val Lys Asn Asn Ile Asp
Lys 275 280 285 Phe
Ile Pro Glu Trp Glu Ile Phe Lys Lys Gln Gly Cys 290
295 300 41861DNABacillus thuringiensis 41atgaataata
ttaataagaa gtatagactt aatgatatga ttaataaaaa tcaatttatt 60atttcaaaaa
cagaatgggt tactataaga acatatattg aaattggatt aactttacca 120gtaaatgaac
aagatttacg aaaatatttc aatttaaatc cagatataac actatctaat 180gatttttctg
aattatttga tatttgttat tctattaaaa atttagctca atggtggaat 240accactatac
ttcctttaat tattaaatct gttaataata ttacatcata tggatttaaa 300attgctggta
atccttttaa taataaagaa ggatactttt caaaattaca aaatgaatta 360catattatta
ataattataa ttctaataaa acaacaaaaa ctattaaaca atttcaatct 420cggtgtaaca
ttttaattaa ggaagttaaa caatatgaag atgttaccaa aaatattgta 480atattattaa
ataaactttt atatggtaat cagcaaaaat tagaagggat tattaatatt 540caaaaacgat
taaaagtggt tcaaacaact tttaatccga tatctaatga aactaattta 600atttataaaa
aactatttga aaaaataaaa aaaataaata ttggtttttt tgaatgcgta 660aataaatata
taagtatatt taacaaaata attattatgt ggtcaaatac tgaacaacaa 720attatagatt
ttaaatcaat tttatttcaa gaatttaaaa atataaatga aacagttatt 780gaatttgaag
atattattga gatttggtta attatagcta aaaaatctcg tgaatttact 840ttaaatgctt
atatatctta g
86142286PRTBacillus thuringiensis 42Met Asn Asn Ile Asn Lys Lys Tyr Arg
Leu Asn Asp Met Ile Asn Lys 1 5 10
15 Asn Gln Phe Ile Ile Ser Lys Thr Glu Trp Val Thr Ile Arg
Thr Tyr 20 25 30
Ile Glu Ile Gly Leu Thr Leu Pro Val Asn Glu Gln Asp Leu Arg Lys
35 40 45 Tyr Phe Asn Leu
Asn Pro Asp Ile Thr Leu Ser Asn Asp Phe Ser Glu 50
55 60 Leu Phe Asp Ile Cys Tyr Ser Ile
Lys Asn Leu Ala Gln Trp Trp Asn 65 70
75 80 Thr Thr Ile Leu Pro Leu Ile Ile Lys Ser Val Asn
Asn Ile Thr Ser 85 90
95 Tyr Gly Phe Lys Ile Ala Gly Asn Pro Phe Asn Asn Lys Glu Gly Tyr
100 105 110 Phe Ser Lys
Leu Gln Asn Glu Leu His Ile Ile Asn Asn Tyr Asn Ser 115
120 125 Asn Lys Thr Thr Lys Thr Ile Lys
Gln Phe Gln Ser Arg Cys Asn Ile 130 135
140 Leu Ile Lys Glu Val Lys Gln Tyr Glu Asp Val Thr Lys
Asn Ile Val 145 150 155
160 Ile Leu Leu Asn Lys Leu Leu Tyr Gly Asn Gln Gln Lys Leu Glu Gly
165 170 175 Ile Ile Asn Ile
Gln Lys Arg Leu Lys Val Val Gln Thr Thr Phe Asn 180
185 190 Pro Ile Ser Asn Glu Thr Asn Leu Ile
Tyr Lys Lys Leu Phe Glu Lys 195 200
205 Ile Lys Lys Ile Asn Ile Gly Phe Phe Glu Cys Val Asn Lys
Tyr Ile 210 215 220
Ser Ile Phe Asn Lys Ile Ile Ile Met Trp Ser Asn Thr Glu Gln Gln 225
230 235 240 Ile Ile Asp Phe Lys
Ser Ile Leu Phe Gln Glu Phe Lys Asn Ile Asn 245
250 255 Glu Thr Val Ile Glu Phe Glu Asp Ile Ile
Glu Ile Trp Leu Ile Ile 260 265
270 Ala Lys Lys Ser Arg Glu Phe Thr Leu Asn Ala Tyr Ile Ser
275 280 285 43861DNAArtificial
sequenceSynthetic 43atgaacaaca ttaacaagaa gtaccgcctg aacgacatga
ttaacaagaa ccagttcatc 60attagcaaga ctgagtgggt gaccatcagg acgtacatcg
agatcggcct gaccctgccc 120gtgaacgagc aagacctgag gaagtacttc aacctcaacc
ctgacattac actgagcaac 180gacttctcgg agctgttcga catctgctac tctatcaaga
atctcgccca atggtggaac 240acaacgatcc ttccgctaat cattaagtct gtgaacaaca
tcactagcta tggcttcaag 300atcgcgggca acccgttcaa caataaggag ggttacttta
gtaagctcca gaacgaactc 360cacatcatta acaattacaa ttcgaacaag acgactaaga
cgatcaagca gtttcagtca 420cggtgcaaca tcttgattaa ggaggttaaa caatacgaag
atgtaacgaa gaacatcgtg 480atcctcctta acaagctcct gtacggcaac cagcagaagc
tcgaagggat cattaacata 540cagaagcgtc tcaaggtggt ccagaccacg ttcaatccca
tctccaatga gactaacctt 600atctacaaga aactctttga gaagatcaag aagatcaaca
tcggcttctt tgagtgtgtc 660aacaagtaca tctccatctt caacaagata atcattatgt
ggtcaaacac tgagcagcag 720atcatcgact tcaagtccat actgttccag gagttcaaga
acatcaatga gaccgtcatc 780gagttcgaag acatcatcga gatttggctt atcatcgcta
agaagtctag ggagttcacc 840ctgaatgcgt acatctcctg a
86144286PRTArtificial sequenceSynthetic 44Met Asn
Asn Ile Asn Lys Lys Tyr Arg Leu Asn Asp Met Ile Asn Lys 1 5
10 15 Asn Gln Phe Ile Ile Ser Lys
Thr Glu Trp Val Thr Ile Arg Thr Tyr 20 25
30 Ile Glu Ile Gly Leu Thr Leu Pro Val Asn Glu Gln
Asp Leu Arg Lys 35 40 45
Tyr Phe Asn Leu Asn Pro Asp Ile Thr Leu Ser Asn Asp Phe Ser Glu
50 55 60 Leu Phe Asp
Ile Cys Tyr Ser Ile Lys Asn Leu Ala Gln Trp Trp Asn 65
70 75 80 Thr Thr Ile Leu Pro Leu Ile
Ile Lys Ser Val Asn Asn Ile Thr Ser 85
90 95 Tyr Gly Phe Lys Ile Ala Gly Asn Pro Phe Asn
Asn Lys Glu Gly Tyr 100 105
110 Phe Ser Lys Leu Gln Asn Glu Leu His Ile Ile Asn Asn Tyr Asn
Ser 115 120 125 Asn
Lys Thr Thr Lys Thr Ile Lys Gln Phe Gln Ser Arg Cys Asn Ile 130
135 140 Leu Ile Lys Glu Val Lys
Gln Tyr Glu Asp Val Thr Lys Asn Ile Val 145 150
155 160 Ile Leu Leu Asn Lys Leu Leu Tyr Gly Asn Gln
Gln Lys Leu Glu Gly 165 170
175 Ile Ile Asn Ile Gln Lys Arg Leu Lys Val Val Gln Thr Thr Phe Asn
180 185 190 Pro Ile
Ser Asn Glu Thr Asn Leu Ile Tyr Lys Lys Leu Phe Glu Lys 195
200 205 Ile Lys Lys Ile Asn Ile Gly
Phe Phe Glu Cys Val Asn Lys Tyr Ile 210 215
220 Ser Ile Phe Asn Lys Ile Ile Ile Met Trp Ser Asn
Thr Glu Gln Gln 225 230 235
240 Ile Ile Asp Phe Lys Ser Ile Leu Phe Gln Glu Phe Lys Asn Ile Asn
245 250 255 Glu Thr Val
Ile Glu Phe Glu Asp Ile Ile Glu Ile Trp Leu Ile Ile 260
265 270 Ala Lys Lys Ser Arg Glu Phe Thr
Leu Asn Ala Tyr Ile Ser 275 280
285 45960DNABacillus thuringiensis 45atgacaatta caaatatcga attagctata
cgagattata caaattggga tggtactcgt 60gaaattccgg gatacatcaa tcgtcaggtt
atagatgggc caaatatata tgactatgta 120attagtgact ctgtagctgt tccaaaaact
gtaattttca atgtaaatcc aactccatat 180accgggccta atataatatc agaaaataat
acagatgtaa atcaaaataa acgtattaag 240ttttctgaaa aagtagttga aactactaca
catactacta caaagggttt taaaattggc 300ggtggaatta aatctactac aaaaggaact
ttaaaattaa aatttcctgt aggagaacta 360gggtttgagc aaactcttga gctacctctt
acaggagaat acaatagtag ttccactacg 420gggaacactt gtgcaaatga aaaattatgg
gaaataacag ataatataac tgtacctcca 480cattcacgtg taacttcaac tttaataatt
atgaaaacgg aagtaagggt tccaatggaa 540ttaaccacta atcttagagg aactaattct
agtggtgaag gctcgttccc tactagtaat 600ggtctttttt catatactac ttcagctcgt
ggaactgtag gcggtatttt tgttagttat 660tacgtgaggc ctgcttctgc attgtataat
acctcttggc ctgataaacc tgcaactttt 720aattctattg gctcaaatga atctctaaat
ttattgggat ctggatattc tgacgtagtt 780ccatctctat atgttactat tagacaagat
caaactccat tatcaggata tccaggtgaa 840acgaaaacct ggtattcaga taaagtgata
ttaagagatg gaagaattgt aacactacca 900agcaatgctg atgtaaatat gtcacaaaca
gccaaaattc catattgtga tagatcttaa 96046319PRTBacillus thuringiensis
46Met Thr Ile Thr Asn Ile Glu Leu Ala Ile Arg Asp Tyr Thr Asn Trp 1
5 10 15 Asp Gly Thr Arg
Glu Ile Pro Gly Tyr Ile Asn Arg Gln Val Ile Asp 20
25 30 Gly Pro Asn Ile Tyr Asp Tyr Val Ile
Ser Asp Ser Val Ala Val Pro 35 40
45 Lys Thr Val Ile Phe Asn Val Asn Pro Thr Pro Tyr Thr Gly
Pro Asn 50 55 60
Ile Ile Ser Glu Asn Asn Thr Asp Val Asn Gln Asn Lys Arg Ile Lys 65
70 75 80 Phe Ser Glu Lys Val
Val Glu Thr Thr Thr His Thr Thr Thr Lys Gly 85
90 95 Phe Lys Ile Gly Gly Gly Ile Lys Ser Thr
Thr Lys Gly Thr Leu Lys 100 105
110 Leu Lys Phe Pro Val Gly Glu Leu Gly Phe Glu Gln Thr Leu Glu
Leu 115 120 125 Pro
Leu Thr Gly Glu Tyr Asn Ser Ser Ser Thr Thr Gly Asn Thr Cys 130
135 140 Ala Asn Glu Lys Leu Trp
Glu Ile Thr Asp Asn Ile Thr Val Pro Pro 145 150
155 160 His Ser Arg Val Thr Ser Thr Leu Ile Ile Met
Lys Thr Glu Val Arg 165 170
175 Val Pro Met Glu Leu Thr Thr Asn Leu Arg Gly Thr Asn Ser Ser Gly
180 185 190 Glu Gly
Ser Phe Pro Thr Ser Asn Gly Leu Phe Ser Tyr Thr Thr Ser 195
200 205 Ala Arg Gly Thr Val Gly Gly
Ile Phe Val Ser Tyr Tyr Val Arg Pro 210 215
220 Ala Ser Ala Leu Tyr Asn Thr Ser Trp Pro Asp Lys
Pro Ala Thr Phe 225 230 235
240 Asn Ser Ile Gly Ser Asn Glu Ser Leu Asn Leu Leu Gly Ser Gly Tyr
245 250 255 Ser Asp Val
Val Pro Ser Leu Tyr Val Thr Ile Arg Gln Asp Gln Thr 260
265 270 Pro Leu Ser Gly Tyr Pro Gly Glu
Thr Lys Thr Trp Tyr Ser Asp Lys 275 280
285 Val Ile Leu Arg Asp Gly Arg Ile Val Thr Leu Pro Ser
Asn Ala Asp 290 295 300
Val Asn Met Ser Gln Thr Ala Lys Ile Pro Tyr Cys Asp Arg Ser 305
310 315 47960DNAArtificial
sequenceSynthetic 47atgaccatca ccaacatcga actcgccatc cgcgactaca
ctaactggga cggcactagg 60gagatccctg gctacatcaa ccgccaagtg atcgacggcc
cgaacatcta cgactacgtg 120atctctgact ccgtggccgt gcctaagacc gtcatcttca
acgtgaaccc aactccgtac 180actgggccaa acatcataag cgagaacaat actgacgtta
atcagaacaa gcgcattaag 240ttctcggaga aggttgtgga gaccactacc catacgacca
caaagggctt caagatcggt 300ggcggtatca agtccaccac gaagggcact ctcaagctga
aattccctgt cggtgagctt 360ggatttgagc aaaccctcga acttcctctg acgggcgagt
acaactccag ctccaccact 420gggaacacct gcgccaacga gaaactgtgg gaaattaccg
acaacataac cgtcccgcca 480cactcccgag tcacctctac gcttatcatt atgaagacag
aagtaagggt gccgatggag 540ttgacgacta acctgcgtgg aacaaacagt tccggcgaag
ggagctttcc tacctctaat 600ggcctcttta gctacaccac atcagctaga ggaaccgttg
gcggtatctt cgtttcttac 660tacgtccgtc cggccagcgc actctacaac acatcatggc
ctgacaagcc cgcgaccttc 720aatagcattg gtagcaatga gtcgctcaac ctgctcggtt
ccggttacag cgatgttgtg 780cccagtctct atgttacgat ccggcaagat cagactccgc
tgtccggcta cccaggcgag 840acaaagacat ggtactccga taaggtcatc cttcgtgatg
ggcgcattgt caccctccct 900tctaatgcgg atgtcaacat gagccagacg gctaagatcc
cgtattgcga ccggtcctga 96048319PRTArtificial sequenceSynthetic 48Met
Thr Ile Thr Asn Ile Glu Leu Ala Ile Arg Asp Tyr Thr Asn Trp 1
5 10 15 Asp Gly Thr Arg Glu Ile
Pro Gly Tyr Ile Asn Arg Gln Val Ile Asp 20
25 30 Gly Pro Asn Ile Tyr Asp Tyr Val Ile Ser
Asp Ser Val Ala Val Pro 35 40
45 Lys Thr Val Ile Phe Asn Val Asn Pro Thr Pro Tyr Thr Gly
Pro Asn 50 55 60
Ile Ile Ser Glu Asn Asn Thr Asp Val Asn Gln Asn Lys Arg Ile Lys 65
70 75 80 Phe Ser Glu Lys Val
Val Glu Thr Thr Thr His Thr Thr Thr Lys Gly 85
90 95 Phe Lys Ile Gly Gly Gly Ile Lys Ser Thr
Thr Lys Gly Thr Leu Lys 100 105
110 Leu Lys Phe Pro Val Gly Glu Leu Gly Phe Glu Gln Thr Leu Glu
Leu 115 120 125 Pro
Leu Thr Gly Glu Tyr Asn Ser Ser Ser Thr Thr Gly Asn Thr Cys 130
135 140 Ala Asn Glu Lys Leu Trp
Glu Ile Thr Asp Asn Ile Thr Val Pro Pro 145 150
155 160 His Ser Arg Val Thr Ser Thr Leu Ile Ile Met
Lys Thr Glu Val Arg 165 170
175 Val Pro Met Glu Leu Thr Thr Asn Leu Arg Gly Thr Asn Ser Ser Gly
180 185 190 Glu Gly
Ser Phe Pro Thr Ser Asn Gly Leu Phe Ser Tyr Thr Thr Ser 195
200 205 Ala Arg Gly Thr Val Gly Gly
Ile Phe Val Ser Tyr Tyr Val Arg Pro 210 215
220 Ala Ser Ala Leu Tyr Asn Thr Ser Trp Pro Asp Lys
Pro Ala Thr Phe 225 230 235
240 Asn Ser Ile Gly Ser Asn Glu Ser Leu Asn Leu Leu Gly Ser Gly Tyr
245 250 255 Ser Asp Val
Val Pro Ser Leu Tyr Val Thr Ile Arg Gln Asp Gln Thr 260
265 270 Pro Leu Ser Gly Tyr Pro Gly Glu
Thr Lys Thr Trp Tyr Ser Asp Lys 275 280
285 Val Ile Leu Arg Asp Gly Arg Ile Val Thr Leu Pro Ser
Asn Ala Asp 290 295 300
Val Asn Met Ser Gln Thr Ala Lys Ile Pro Tyr Cys Asp Arg Ser 305
310 315 49855DNABacillus
thuringiensis 49atgaaatatc acaaaaaatt tcatgaaata gcttgggagt ttgctgaaaa
atggactgaa 60caagaaggtt tggagttagc aaatgtcgat tatgtgaatc ctgctactgg
taaagacacg 120ttgaatttcg tgaataaatt tgaatatatc ggaaaaataa aagagggaaa
tctttgccca 180gagatagtag caaatgagtc tttttcaaat tcaaaatgtg atactttgaa
aaataatctc 240cataaaaaat tatttttgaa acaatattat ttatgggata tagactatca
atttataatt 300ccgccttcaa tttatacaaa tccaatatta ccgccatgtc ttagtaaaaa
aatcaatcca 360gcaatacagg tagatttatt taaaagagca taccattttg aatctcaact
aaacaattct 420gaaataatag aagcagggat ttatattgaa cctaatcaaa cgataaatgc
taaagtaata 480gcagaatata aaaatgtgca acaaaaatat tgtatacacc ttaaaatttc
aggaagtatt 540gttattgaag tgaaaaaaaa tcgtaattct tgtaaggatt ctaaaacatt
ttatactatc 600ccaatcgtag atttgtataa atcagagctt gcacataatc attcttttca
tttagatggg 660gagactgtta tatttactga aaaaggtatg tttaaaggcc taatttgttc
taatgtattt 720atcgaagggg aacggtttaa tttaaaaaca ggagaatgct taggtaaata
tataatacca 780ttaggtatgg acgaagaaaa agttctagaa aatagtaaat caatattttt
taattcagaa 840aaaggaggaa tttaa
85550284PRTBacillus thuringiensis 50Met Lys Tyr His Lys Lys
Phe His Glu Ile Ala Trp Glu Phe Ala Glu 1 5
10 15 Lys Trp Thr Glu Gln Glu Gly Leu Glu Leu Ala
Asn Val Asp Tyr Val 20 25
30 Asn Pro Ala Thr Gly Lys Asp Thr Leu Asn Phe Val Asn Lys Phe
Glu 35 40 45 Tyr
Ile Gly Lys Ile Lys Glu Gly Asn Leu Cys Pro Glu Ile Val Ala 50
55 60 Asn Glu Ser Phe Ser Asn
Ser Lys Cys Asp Thr Leu Lys Asn Asn Leu 65 70
75 80 His Lys Lys Leu Phe Leu Lys Gln Tyr Tyr Leu
Trp Asp Ile Asp Tyr 85 90
95 Gln Phe Ile Ile Pro Pro Ser Ile Tyr Thr Asn Pro Ile Leu Pro Pro
100 105 110 Cys Leu
Ser Lys Lys Ile Asn Pro Ala Ile Gln Val Asp Leu Phe Lys 115
120 125 Arg Ala Tyr His Phe Glu Ser
Gln Leu Asn Asn Ser Glu Ile Ile Glu 130 135
140 Ala Gly Ile Tyr Ile Glu Pro Asn Gln Thr Ile Asn
Ala Lys Val Ile 145 150 155
160 Ala Glu Tyr Lys Asn Val Gln Gln Lys Tyr Cys Ile His Leu Lys Ile
165 170 175 Ser Gly Ser
Ile Val Ile Glu Val Lys Lys Asn Arg Asn Ser Cys Lys 180
185 190 Asp Ser Lys Thr Phe Tyr Thr Ile
Pro Ile Val Asp Leu Tyr Lys Ser 195 200
205 Glu Leu Ala His Asn His Ser Phe His Leu Asp Gly Glu
Thr Val Ile 210 215 220
Phe Thr Glu Lys Gly Met Phe Lys Gly Leu Ile Cys Ser Asn Val Phe 225
230 235 240 Ile Glu Gly Glu
Arg Phe Asn Leu Lys Thr Gly Glu Cys Leu Gly Lys 245
250 255 Tyr Ile Ile Pro Leu Gly Met Asp Glu
Glu Lys Val Leu Glu Asn Ser 260 265
270 Lys Ser Ile Phe Phe Asn Ser Glu Lys Gly Gly Ile
275 280 51855DNAArtificial
sequenceSynthetic 51atgaagtacc acaagaagtt ccacgagatc gcctgggagt
tcgctgagaa gtggactgag 60caggagggcc tagagctggc taacgtggac tacgtgaacc
ctgccaccgg caaggacacc 120ctgaacttcg tgaacaagtt cgagtacatc ggaaagatta
aggaggggaa cctctgtccc 180gagattgttg ccaatgagtc tttcagcaat agcaagtgcg
atacgctcaa gaataacctc 240cacaagaaac tgttcttgaa gcagtactac ctgtgggaca
tcgactacca gttcatcatt 300ccaccgagca tctacaccaa ccctatcctc cctccatgcc
tgtcgaagaa gatcaacccg 360gccatccaag tggacctctt caagcgcgct taccactttg
agtcccagct taacaatagc 420gagatcatcg aggccggtat ctacattgaa cccaaccaga
ccatcaatgc gaaggttatc 480gcggagtaca agaacgtgca acagaagtat tgcatccacc
tcaagatcag tggtagtatt 540gtcatcgagg tcaagaagaa caggaactcg tgcaaggact
caaagacgtt ctacactatc 600ccgatagtgg acctttacaa gtctgaactc gcccacaacc
attcattcca tctcgatgga 660gagactgtaa tcttcaccga gaagggcatg tttaagggcc
ttatttgctc caacgtcttc 720atcgaaggcg aacgcttcaa ccttaagaca ggagaatgcc
tggggaagta catcatccct 780ttgggcatgg atgaggagaa ggttctggag aactccaagt
ctatcttctt caactccgag 840aagggcggga tctga
85552284PRTArtificial sequenceSynthetic 52Met Lys
Tyr His Lys Lys Phe His Glu Ile Ala Trp Glu Phe Ala Glu 1 5
10 15 Lys Trp Thr Glu Gln Glu Gly
Leu Glu Leu Ala Asn Val Asp Tyr Val 20 25
30 Asn Pro Ala Thr Gly Lys Asp Thr Leu Asn Phe Val
Asn Lys Phe Glu 35 40 45
Tyr Ile Gly Lys Ile Lys Glu Gly Asn Leu Cys Pro Glu Ile Val Ala
50 55 60 Asn Glu Ser
Phe Ser Asn Ser Lys Cys Asp Thr Leu Lys Asn Asn Leu 65
70 75 80 His Lys Lys Leu Phe Leu Lys
Gln Tyr Tyr Leu Trp Asp Ile Asp Tyr 85
90 95 Gln Phe Ile Ile Pro Pro Ser Ile Tyr Thr Asn
Pro Ile Leu Pro Pro 100 105
110 Cys Leu Ser Lys Lys Ile Asn Pro Ala Ile Gln Val Asp Leu Phe
Lys 115 120 125 Arg
Ala Tyr His Phe Glu Ser Gln Leu Asn Asn Ser Glu Ile Ile Glu 130
135 140 Ala Gly Ile Tyr Ile Glu
Pro Asn Gln Thr Ile Asn Ala Lys Val Ile 145 150
155 160 Ala Glu Tyr Lys Asn Val Gln Gln Lys Tyr Cys
Ile His Leu Lys Ile 165 170
175 Ser Gly Ser Ile Val Ile Glu Val Lys Lys Asn Arg Asn Ser Cys Lys
180 185 190 Asp Ser
Lys Thr Phe Tyr Thr Ile Pro Ile Val Asp Leu Tyr Lys Ser 195
200 205 Glu Leu Ala His Asn His Ser
Phe His Leu Asp Gly Glu Thr Val Ile 210 215
220 Phe Thr Glu Lys Gly Met Phe Lys Gly Leu Ile Cys
Ser Asn Val Phe 225 230 235
240 Ile Glu Gly Glu Arg Phe Asn Leu Lys Thr Gly Glu Cys Leu Gly Lys
245 250 255 Tyr Ile Ile
Pro Leu Gly Met Asp Glu Glu Lys Val Leu Glu Asn Ser 260
265 270 Lys Ser Ile Phe Phe Asn Ser Glu
Lys Gly Gly Ile 275 280
53903DNABacillus thuringiensis 53atgctagcat tcatattttc aggaggaagt
agccaacttt gtacgtcaat acgtaatgaa 60tataataaca tattgcaagg gggggggatg
gatgtatctt catccttccg tgtagatcct 120tctaaaataa atataaatga tttggaagtg
acatatccag agttaaatat tcccgataac 180ataatagctt ctatagtatc taataatagc
ttcgagaata gaggatttat aactaacgaa 240acttacgcta ccgctgaggt aaccagagca
ttaaccgaaa caattactac agctactact 300agagggttta aattcacgca aggttttacc
tatacaaata agattaacct cagaatacca 360attgccggtt cagaatcaac aatttccttt
tcaacatctt ttgaacaaaa tatttcaacc 420actgaaacaa taacaaaaac tgaaacgata
actatacttg tacctagaca aacggttaca 480gtaaggccta gaacaagaaa agttgttcaa
ataagacttt atcaattacg aattcctaga 540gtattcaccg aaatatctgc ttatgtaaca
ggtactctta gacaaccaat tcctcaagcg 600aatcctgatg tttatgctac tctggtaagt
gtcaataatg catgtcctaa tgcatttgtt 660aaccgtgaca attttttgag aatagatcgc
gaaaagcggg gcttagcgtt aagaggagaa 720ggtgaattta gtggaaatat agtttcatta
gatttcctga ttacgactac tgaatatgat 780ttggatacaa atgctattat taatatagat
aataccttgg gcagagcagc tattctagga 840agtgagcctt gtggaactat tgcttataca
gaaccaattg acattattca cactgattgt 900taa
90354300PRTBacillus thuringiensis
54Met Leu Ala Phe Ile Phe Ser Gly Gly Ser Ser Gln Leu Cys Thr Ser 1
5 10 15 Ile Arg Asn Glu
Tyr Asn Asn Ile Leu Gln Gly Gly Gly Met Asp Val 20
25 30 Ser Ser Ser Phe Arg Val Asp Pro Ser
Lys Ile Asn Ile Asn Asp Leu 35 40
45 Glu Val Thr Tyr Pro Glu Leu Asn Ile Pro Asp Asn Ile Ile
Ala Ser 50 55 60
Ile Val Ser Asn Asn Ser Phe Glu Asn Arg Gly Phe Ile Thr Asn Glu 65
70 75 80 Thr Tyr Ala Thr Ala
Glu Val Thr Arg Ala Leu Thr Glu Thr Ile Thr 85
90 95 Thr Ala Thr Thr Arg Gly Phe Lys Phe Thr
Gln Gly Phe Thr Tyr Thr 100 105
110 Asn Lys Ile Asn Leu Arg Ile Pro Ile Ala Gly Ser Glu Ser Thr
Ile 115 120 125 Ser
Phe Ser Thr Ser Phe Glu Gln Asn Ile Ser Thr Thr Glu Thr Ile 130
135 140 Thr Lys Thr Glu Thr Ile
Thr Ile Leu Val Pro Arg Gln Thr Val Thr 145 150
155 160 Val Arg Pro Arg Thr Arg Lys Val Val Gln Ile
Arg Leu Tyr Gln Leu 165 170
175 Arg Ile Pro Arg Val Phe Thr Glu Ile Ser Ala Tyr Val Thr Gly Thr
180 185 190 Leu Arg
Gln Pro Ile Pro Gln Ala Asn Pro Asp Val Tyr Ala Thr Leu 195
200 205 Val Ser Val Asn Asn Ala Cys
Pro Asn Ala Phe Val Asn Arg Asp Asn 210 215
220 Phe Leu Arg Ile Asp Arg Glu Lys Arg Gly Leu Ala
Leu Arg Gly Glu 225 230 235
240 Gly Glu Phe Ser Gly Asn Ile Val Ser Leu Asp Phe Leu Ile Thr Thr
245 250 255 Thr Glu Tyr
Asp Leu Asp Thr Asn Ala Ile Ile Asn Ile Asp Asn Thr 260
265 270 Leu Gly Arg Ala Ala Ile Leu Gly
Ser Glu Pro Cys Gly Thr Ile Ala 275 280
285 Tyr Thr Glu Pro Ile Asp Ile Ile His Thr Asp Cys
290 295 300 55903DNAArtificial
sequenceSynthetic 55atgcttgcct tcatcttctc cggcggtagc tcccagctct
gcactagcat ccgtaacgag 60tacaacaaca tcctccaggg cggtggcatg gacgtcagct
ccagcttccg cgtggaccct 120agcaagatca acatcaacga ccttgaggtg acctatcctg
agttgaacat tccagataac 180attatcgcta gtattgtttc caacaacagt ttcgagaaca
ggggattcat cactaacgag 240acttacgcca cggccgaagt aacccgcgct cttactgaga
cgattacgac tgccaccacg 300cgcggattca agtttaccca ggggttcacc tacaccaaca
agatcaacct ccgcatccca 360atcgctggct ctgagtccac catctccttc tctacgtcgt
tcgaacagaa catctcgacc 420actgagacca tcacaaagac cgagactatc accatccttg
tgccaaggca gaccgtcacc 480gtcagacctc ggaccaggaa ggtcgtgcaa atccgtcttt
atcagttgcg tattccgagg 540gtgttcaccg agatttcagc gtatgttact ggcactctcc
ggcagccgat ccctcaagca 600aacccggacg tttacgctac ccttgtttcc gtcaacaatg
cctgccccaa tgctttcgtg 660aaccgagaca acttccttcg catcgaccgg gagaagcgcg
gtctcgcgct gcgtggcgag 720ggcgagttct ctggaaacat cgtctccctg gatttcctga
ttactacaac ggagtacgat 780ctggatacaa acgctatcat caacattgac aacacgctcg
ggcgcgcggc catacttggg 840tcggaaccct gcggtacaat agcatacaca gagcccatcg
acatcattca cacagattgt 900tga
90356300PRTArtificial sequenceSynthetic 56Met Leu
Ala Phe Ile Phe Ser Gly Gly Ser Ser Gln Leu Cys Thr Ser 1 5
10 15 Ile Arg Asn Glu Tyr Asn Asn
Ile Leu Gln Gly Gly Gly Met Asp Val 20 25
30 Ser Ser Ser Phe Arg Val Asp Pro Ser Lys Ile Asn
Ile Asn Asp Leu 35 40 45
Glu Val Thr Tyr Pro Glu Leu Asn Ile Pro Asp Asn Ile Ile Ala Ser
50 55 60 Ile Val Ser
Asn Asn Ser Phe Glu Asn Arg Gly Phe Ile Thr Asn Glu 65
70 75 80 Thr Tyr Ala Thr Ala Glu Val
Thr Arg Ala Leu Thr Glu Thr Ile Thr 85
90 95 Thr Ala Thr Thr Arg Gly Phe Lys Phe Thr Gln
Gly Phe Thr Tyr Thr 100 105
110 Asn Lys Ile Asn Leu Arg Ile Pro Ile Ala Gly Ser Glu Ser Thr
Ile 115 120 125 Ser
Phe Ser Thr Ser Phe Glu Gln Asn Ile Ser Thr Thr Glu Thr Ile 130
135 140 Thr Lys Thr Glu Thr Ile
Thr Ile Leu Val Pro Arg Gln Thr Val Thr 145 150
155 160 Val Arg Pro Arg Thr Arg Lys Val Val Gln Ile
Arg Leu Tyr Gln Leu 165 170
175 Arg Ile Pro Arg Val Phe Thr Glu Ile Ser Ala Tyr Val Thr Gly Thr
180 185 190 Leu Arg
Gln Pro Ile Pro Gln Ala Asn Pro Asp Val Tyr Ala Thr Leu 195
200 205 Val Ser Val Asn Asn Ala Cys
Pro Asn Ala Phe Val Asn Arg Asp Asn 210 215
220 Phe Leu Arg Ile Asp Arg Glu Lys Arg Gly Leu Ala
Leu Arg Gly Glu 225 230 235
240 Gly Glu Phe Ser Gly Asn Ile Val Ser Leu Asp Phe Leu Ile Thr Thr
245 250 255 Thr Glu Tyr
Asp Leu Asp Thr Asn Ala Ile Ile Asn Ile Asp Asn Thr 260
265 270 Leu Gly Arg Ala Ala Ile Leu Gly
Ser Glu Pro Cys Gly Thr Ile Ala 275 280
285 Tyr Thr Glu Pro Ile Asp Ile Ile His Thr Asp Cys
290 295 300 57597DNABacillus
thuringiensis 57atgaatacgt atcataaaat atgttctggt ccttatcagt ataatcacaa
aggtccctac 60acggaatgtg ggcctactcc taggagatcc aatcccgatg aagaacattc
aactttaaaa 120gattcatacg gtgatcctgt cctctattac atgccctatt atatggatcc
atatgaattt 180cctggtgaaa gatttgtgta cgaatggact tctgatggag atcgtggagt
taaattagga 240acgtcttatg cgaaagttta tttgcacttt tggaaagcta atggttccga
tagtgcttta 300cgtatcaata tattggatcg tgagggaaac ccagaaaatc agtacttatc
taagaaacgt 360ggtactgacg aagtactatt atataatcat gaccctctag aatccgaatg
ggttccggag 420aattcatctg atcctaattt ttcagtagga aactacttct cactccaaaa
tgcttcggat 480catattattt ccaatgaaaa attcttatcc tatagcattc caggtgaatg
gttaaccaca 540gttcagccta ctatgaattc aaaaacaatg tggcgtttaa ttccaacatg
gaaataa 59758198PRTBacillus thuringiensis 58Met Asn Thr Tyr His Lys
Ile Cys Ser Gly Pro Tyr Gln Tyr Asn His 1 5
10 15 Lys Gly Pro Tyr Thr Glu Cys Gly Pro Thr Pro
Arg Arg Ser Asn Pro 20 25
30 Asp Glu Glu His Ser Thr Leu Lys Asp Ser Tyr Gly Asp Pro Val
Leu 35 40 45 Tyr
Tyr Met Pro Tyr Tyr Met Asp Pro Tyr Glu Phe Pro Gly Glu Arg 50
55 60 Phe Val Tyr Glu Trp Thr
Ser Asp Gly Asp Arg Gly Val Lys Leu Gly 65 70
75 80 Thr Ser Tyr Ala Lys Val Tyr Leu His Phe Trp
Lys Ala Asn Gly Ser 85 90
95 Asp Ser Ala Leu Arg Ile Asn Ile Leu Asp Arg Glu Gly Asn Pro Glu
100 105 110 Asn Gln
Tyr Leu Ser Lys Lys Arg Gly Thr Asp Glu Val Leu Leu Tyr 115
120 125 Asn His Asp Pro Leu Glu Ser
Glu Trp Val Pro Glu Asn Ser Ser Asp 130 135
140 Pro Asn Phe Ser Val Gly Asn Tyr Phe Ser Leu Gln
Asn Ala Ser Asp 145 150 155
160 His Ile Ile Ser Asn Glu Lys Phe Leu Ser Tyr Ser Ile Pro Gly Glu
165 170 175 Trp Leu Thr
Thr Val Gln Pro Thr Met Asn Ser Lys Thr Met Trp Arg 180
185 190 Leu Ile Pro Thr Trp Lys
195 59660DNABacillus thuringiensis 59atgaatacgc atcataaaat
gtgttctagt ccttatcgat ataatcatag acataaggat 60tgcgattgtc atttatataa
ccataacggt ccttatcagt ataatcacag aggtccctac 120acggaatgtg gacctactcc
taggagatca aatcccgatg aagaacattc aactttaaaa 180gattcatacg gtgatccagt
ccgctatagc atgccctatt atatggatcc atatgaattt 240cctggtgaaa gatttgtgta
cgaatggact tctgatggag atcgtggagt taaattagga 300acgtcttatg cgagtgttta
tttgtacttt tggaaaatta ataatggtac cgatagtgct 360ttacgtatca atatattgga
tcgtgaggga aacccagaaa atcagtactt atctaagaaa 420cgtggtactg acgaagtact
attatataat cgtgaccctc tagaatccga atgggttccg 480gagaattcat ctgatcctaa
tttttcagta ggaaactact tctcactcca aagtgcttcg 540gatcatatta tttccaatga
aaaattctta tcctatagca ttcccggtga atggttaacc 600acagttcagc ctactatgaa
ttcaaaaaca atgtggcgtt taattccagc atggaaataa 66060219PRTBacillus
thuringiensis 60Met Asn Thr His His Lys Met Cys Ser Ser Pro Tyr Arg Tyr
Asn His 1 5 10 15
Arg His Lys Asp Cys Asp Cys His Leu Tyr Asn His Asn Gly Pro Tyr
20 25 30 Gln Tyr Asn His Arg
Gly Pro Tyr Thr Glu Cys Gly Pro Thr Pro Arg 35
40 45 Arg Ser Asn Pro Asp Glu Glu His Ser
Thr Leu Lys Asp Ser Tyr Gly 50 55
60 Asp Pro Val Arg Tyr Ser Met Pro Tyr Tyr Met Asp Pro
Tyr Glu Phe 65 70 75
80 Pro Gly Glu Arg Phe Val Tyr Glu Trp Thr Ser Asp Gly Asp Arg Gly
85 90 95 Val Lys Leu Gly
Thr Ser Tyr Ala Ser Val Tyr Leu Tyr Phe Trp Lys 100
105 110 Ile Asn Asn Gly Thr Asp Ser Ala Leu
Arg Ile Asn Ile Leu Asp Arg 115 120
125 Glu Gly Asn Pro Glu Asn Gln Tyr Leu Ser Lys Lys Arg Gly
Thr Asp 130 135 140
Glu Val Leu Leu Tyr Asn Arg Asp Pro Leu Glu Ser Glu Trp Val Pro 145
150 155 160 Glu Asn Ser Ser Asp
Pro Asn Phe Ser Val Gly Asn Tyr Phe Ser Leu 165
170 175 Gln Ser Ala Ser Asp His Ile Ile Ser Asn
Glu Lys Phe Leu Ser Tyr 180 185
190 Ser Ile Pro Gly Glu Trp Leu Thr Thr Val Gln Pro Thr Met Asn
Ser 195 200 205 Lys
Thr Met Trp Arg Leu Ile Pro Ala Trp Lys 210 215
61597DNAArtificial sequenceSynthetic 61atgaacacct accacaagat
ttgctccggc ccttaccagt acaaccacaa gggaccgtac 60actgagtgcg gccctacccc
taggcgtagc aaccctgacg aggagcactc cacccttaag 120gactcctacg gtgacccagt
gctctactac atgccgtact acatggaccc atacgagttc 180cctggcgaaa ggttcgtgta
cgagtggacg tcagatggcg atagaggagt caagctcggt 240actagctatg cgaaagttta
cctacacttc tggaaggcta acgggtctga ctccgccttg 300cggatcaaca ttctcgatag
ggagggcaac cctgagaacc agtatctctc caagaagcgc 360ggcaccgatg aggttcttct
gtacaaccac gacccacttg agtccgaatg ggttccagaa 420aacagctcgg acccgaactt
cagcgtggga aactacttca gtctccagaa cgcatcagat 480cacatcatct cgaatgagaa
gtttctctct tattctatcc caggcgagtg gctgactacc 540gtgcaaccta cgatgaatag
taagacaatg tggaggctca ttccgacatg gaagtga 59762198PRTArtificial
sequenceSynthetic 62Met Asn Thr Tyr His Lys Ile Cys Ser Gly Pro Tyr Gln
Tyr Asn His 1 5 10 15
Lys Gly Pro Tyr Thr Glu Cys Gly Pro Thr Pro Arg Arg Ser Asn Pro
20 25 30 Asp Glu Glu His
Ser Thr Leu Lys Asp Ser Tyr Gly Asp Pro Val Leu 35
40 45 Tyr Tyr Met Pro Tyr Tyr Met Asp Pro
Tyr Glu Phe Pro Gly Glu Arg 50 55
60 Phe Val Tyr Glu Trp Thr Ser Asp Gly Asp Arg Gly Val
Lys Leu Gly 65 70 75
80 Thr Ser Tyr Ala Lys Val Tyr Leu His Phe Trp Lys Ala Asn Gly Ser
85 90 95 Asp Ser Ala Leu
Arg Ile Asn Ile Leu Asp Arg Glu Gly Asn Pro Glu 100
105 110 Asn Gln Tyr Leu Ser Lys Lys Arg Gly
Thr Asp Glu Val Leu Leu Tyr 115 120
125 Asn His Asp Pro Leu Glu Ser Glu Trp Val Pro Glu Asn Ser
Ser Asp 130 135 140
Pro Asn Phe Ser Val Gly Asn Tyr Phe Ser Leu Gln Asn Ala Ser Asp 145
150 155 160 His Ile Ile Ser Asn
Glu Lys Phe Leu Ser Tyr Ser Ile Pro Gly Glu 165
170 175 Trp Leu Thr Thr Val Gln Pro Thr Met Asn
Ser Lys Thr Met Trp Arg 180 185
190 Leu Ile Pro Thr Trp Lys 195
63660DNAArtificial sequenceSynthetic 63atgaacaccc accacaagat gtgctctagc
ccttaccgct acaaccaccg ccacaaggac 60tgcgactgcc acctctacaa ccacaacgga
ccctaccagt acaaccatag gggcccttac 120actgagtgtg gccctactcc taggcgtagc
aacccggacg aggaacacag caccctcaag 180gactcctacg gcgaccctgt gagatactcc
atgccttact acatggaccc ttacgagttc 240ccaggcgagc gcttcgtgta cgagtggacc
tccgacggcg acagaggtgt gaagctcggc 300acctcctacg cctccgtgta cctctacttc
tggaagatca acaatggtac tgattccgct 360cttcggatca acatcctgga tagggagggc
aacccggaga accagtatct gagcaagaaa 420cgcggcactg atgaggtgct gctttacaac
cgtgatccgc tggagagcga gtgggtgccg 480gagaactctt cggaccctaa cttcagtgtt
ggaaattact tcagtctcca gtcagcgtca 540gatcacatca tctctaatga gaagttcctg
tcctactcga tcccaggaga atggctcacg 600acagttcaac ccacgatgaa ctcgaagaca
atgtggcgat tgataccagc atggaaatga 66064219PRTArtificial sequencean
amino acid sequence translation of SEQ ID NO 63. 64Met Asn Thr His
His Lys Met Cys Ser Ser Pro Tyr Arg Tyr Asn His 1 5
10 15 Arg His Lys Asp Cys Asp Cys His Leu
Tyr Asn His Asn Gly Pro Tyr 20 25
30 Gln Tyr Asn His Arg Gly Pro Tyr Thr Glu Cys Gly Pro Thr
Pro Arg 35 40 45
Arg Ser Asn Pro Asp Glu Glu His Ser Thr Leu Lys Asp Ser Tyr Gly 50
55 60 Asp Pro Val Arg Tyr
Ser Met Pro Tyr Tyr Met Asp Pro Tyr Glu Phe 65 70
75 80 Pro Gly Glu Arg Phe Val Tyr Glu Trp Thr
Ser Asp Gly Asp Arg Gly 85 90
95 Val Lys Leu Gly Thr Ser Tyr Ala Ser Val Tyr Leu Tyr Phe Trp
Lys 100 105 110 Ile
Asn Asn Gly Thr Asp Ser Ala Leu Arg Ile Asn Ile Leu Asp Arg 115
120 125 Glu Gly Asn Pro Glu Asn
Gln Tyr Leu Ser Lys Lys Arg Gly Thr Asp 130 135
140 Glu Val Leu Leu Tyr Asn Arg Asp Pro Leu Glu
Ser Glu Trp Val Pro 145 150 155
160 Glu Asn Ser Ser Asp Pro Asn Phe Ser Val Gly Asn Tyr Phe Ser Leu
165 170 175 Gln Ser
Ala Ser Asp His Ile Ile Ser Asn Glu Lys Phe Leu Ser Tyr 180
185 190 Ser Ile Pro Gly Glu Trp Leu
Thr Thr Val Gln Pro Thr Met Asn Ser 195 200
205 Lys Thr Met Trp Arg Leu Ile Pro Ala Trp Lys
210 215
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