Modified Cry1Ca toxins useful for controlling insect pests

Abstract

The subject invention relates to modified Cry1Ca insecticidal toxins of Bacillus thuringiensis and polynucleotide sequences encoding these toxins. The use of these toxins in transgenic plants and methods for protecting crops from insect pests are described.

Description

[0001] This application is a divisional application of the invention patent application with application number 201580071104.4 filed on December 16, 2015, and invention name “Modified Cry1Ca toxins useful for controlling insect pests”.

[0002] public domain

[0003] The subject invention relates to modifications of Bacillus thuringiensis insecticidal toxins, polynucleotide sequences encoding these toxins, and transgenic plants that produce these toxins. Background of the Invention

[0004] Insects and other pests cost farmers billions of dollars annually in crop losses and the expense of controlling these pests. In addition to field crop losses, insect pests also burden vegetable and fruit growers, ornamental flower producers, and home gardeners. Losses caused by insect pests in agricultural production settings include reduced crop yields, decreased crop quality, and increased harvest costs.

[0005] Insect pests are mainly controlled by intensive application of chemical pesticides, which work by inhibiting insect growth, preventing insects from feeding or breeding, or causing death. Therefore, good insect control can be achieved, but these chemicals can sometimes also affect other beneficial insects. Another problem derived from the widespread use of chemical pesticides is the emergence of insect resistance colonies. This has been partially alleviated by various resistance management practices, but the demand for alternative pest control agents is increasing. Biological pest control agents, such as Bacillus thuringiensis (Bt) strains expressing insecticidal toxins such as δ-endotoxins, have also been applied to crop plants to produce satisfactory results, providing an alternative or supplement to chemical pesticides. Genes encoding some of these δ-endotoxins have been isolated, and their expression in heterologous hosts has been shown to provide another tool for controlling economically important insect pests. In particular, the expression of insecticidal toxins, such as Bacillus thuringiensis delta-endotoxins, in transgenic plants has provided effective protection against selected insect pests, and transgenic plants expressing such toxins have been commercialized, allowing farmers to reduce the use of chemical insect control agents.

[0006] Lepidoptera is a group of important agricultural, horticultural, and household pests that cause a great deal of damage each year. This order includes larvae and adults that feed on leaves and roots. Lepidopteran pests include, but are not limited to, Achoroia grisea, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon (black cutworm "BCW"), Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsiage mmatalis (spiny bean caterpillar), Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx moth (Bombyx spp.), and many more. mori), Bucculatrix thurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Coliasurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmia feneralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella,Southern corn rootworm ("SWCB"), Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia elutella, Erannistilaria, Estigmene acrea, Eulia salubricola, Eupocoellia ambiguella, Eupocilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea,corn earworm ("CEW")), Heliothis virescens (Tobacco armyworm "TBW"), Hemileuca oliviae, Homoeosoma electellum, Hyphantia cunea, Keiferia lycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra configurata, Manduca hornworm quinquemaculata), Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis (European corn borer "ECB"), Paleacrita vernata (Apple looper), Papiapema nebris (common stem borer), Papiliocres phontes (Giant Swallowtail), Pectinophora gossypiella (Red bell moth), Phryganidia californica (Californica), Phyllonorycter blancardella (Spotted leafminer), Pieris napi (Dark-veined cabbage butterfly), Pieris rapae (Rabbit butterfly), Plathypena scabra (Alfalfa armyworm), Platynota flouendana, Dutch pink moth (Platynota stultana), American artichoke moth (Platyptilia carduidactyla), Indian grain moth (Plodia interpunctella), diamondback moth (Plutella xylostella,diamondback moth (DBM), Pontia protodice (American white butterfly), Pseudaletia unipuncta (white-spotted armyworm), Pseudoplasia includens (soybean looper "SBL"), Sabulodes aegrotata, Schizura concinna (red mountain boat moth), Sitotroga cerealella (wheat moth), Spilonta ocellana (apple moth), Spodoptera eridania (southern armyworm "SAW"), Spodoptera frugiperda (fall armyworm "FAW"), Spodoptera exigua (beet armyworm "BAW"), Thaurnstopoea pityocampa, Ensola bisselliella, Trichoplusia ni (cabbage looper "CL"), Udea rubigalis), Xylomyges curiails, and Yponomeutapadella. Any of the above genera (and others), in general, may also form part of the present invention as targets. Any other insects in these genera (as targets) are also included within the scope of the present invention.

[0007] Bacillus thuringiensis (Bt) is a soil-borne, Gram-positive, spore-forming bacterium that produces insecticidal crystalline proteins known as delta-endotoxins or Cry proteins (reviewed in Schnepf et al., 1998). Novel crystalline (Cry) proteins with novel insecticidal properties continue to be discovered at an increasing rate, and over 440 Cry genes have been reported. Currently, over 450 unique Cry and cytotoxin (Cyt) proteins have been classified into 57 homology classes. Cry proteins are named based on the degree of sequence identity, with primary, secondary, and tertiary boundaries at approximately 45%, 78%, and 95% identity, respectively; closely related alleles are assigned new quaternary designations (Crickmore et al., 1998). A large list of delta-endotoxins is maintained at http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / intro.html and is updated regularly. There are currently 73 major groups of "Cry" toxins (Cry1-Cry73), as well as additional Cyt toxins and plant insecticidal protein (VIP) toxins. Many of these numbered groups contain subgroups designated by capital letters, which in turn have lowercase subgroups (e.g., Cry1 has AL, Cry1A has ai).

[0008] To date, insect-resistant transgenic plants produced using Bt proteins have been successfully registered, deregulated, or commercialized. These include Cry1Ab, Cry1Ac, Cry1F, Vip3A, Cry34Ab1 / Cry35Ab1, and Cry3Bb in maize, Cry1Ac, Vip3A, and Cry2Ab in cotton, and Cry3A in potato. Bt toxins account for over 90% of the biopesticide market, and the genes used in transgenic crops developed to confer resistance to insect feeding are essentially all derived from Bt toxins.

[0009] Cry proteins are orally administered toxicants that act in the midgut cells of susceptible insects. The active forms of many Cry proteins contain three distinct protein domains. The most studied Bt proteins are members of the three-domain Cry delta-endotoxin family. These proteins range in size from approximately 70 kDa to 130 kDa. Analysis of the original protein sequence revealed five highly conserved sequence blocks, as well as a high degree of sequence variability between conserved blocks three and five (Schnepf et al., 1998).

[0010] The three-dimensional crystal structures of Cry1Aa1, Cry2Aa1, Cry3Aa1, Cry3Bb1, Cry4Aa, Cry4Ba, and Cry8Ea1 have been determined. These structures are very similar and consist of three distinct domains with the following characteristics (see de Maagd et al., 2003). Domain I is a bundle of seven α-helices, with helix five surrounded by six amphipathic helices. Evidence suggests that this domain is involved in midgut membrane insertion and pore formation. It shares homology with other pore-forming proteins, including hemolysins and colicins. Domain II consists of three antiparallel β-sheets packaged together in a β-prism. This domain shares homology with certain carbohydrate-binding proteins, including vitelline and jacaline. The loops of this domain play an important role in binding insect midgut receptors. In Cry1A proteins, surface-exposed loops at the vertices of the domain II β-sheet are involved in binding to the lepidopteran cadherin receptor. Domain III is a β-sandwich structure that interacts with another receptor. Examples of such receptors, in the case of the Cry1A protein, include aminopeptidases and alkaline phosphatases (Piggot and Ellar, 2007). Structurally, this domain is related to the sugar-binding domains of proteins such as glucanases, galactose oxidases, and sialidases. This domain binds to certain types of receptor proteins and may be involved in the insertion of oligomeric toxin precursors. Conserved Bt sequence blocks 2 and 3 are located near the N-terminus and C-terminus of domain 2, respectively. Therefore, these conserved sequence blocks 2 and 3 are approximate boundary regions between the three functional domains. These conserved regions, which share DNA and protein homology, have been developed for engineering recombinant Bt toxins (U.S. Patent No. 6,090,931, WO 91 / 01087, WO 95 / 06730, WO 1998022595).

[0011] One of the proposed models for the mode of action of Cry proteins is based on pore formation in the midgut membrane of sensitive insects (Knowlesand Ellar, 1987). In the current version of the model (Bravo et al., 2007), binding to cadherin and aminopeptidase receptors on the midgut membrane of Lepidoptera is essential for Cry protein toxicity. According to the pore formation model, Cry protein poisoning involves several steps: 1) soluble Cry protoxin is proteolytically processed into an activated core toxin; 2) Cry protein binds to the cadherin receptor on the insect midgut; 3) further proteolytic cleavage at the N-terminus of the core toxin to remove the α-helical region; 4) Cry protein oligomerization forms a front pore; 5) the front pore binds to the second site membrane receptor (aminopeptidase and alkaline phosphatase); 6) the front pore is inserted into the membrane; and 7) osmotic cell lysis causes midgut rupture and insect death.

[0012] With the widespread use of insect-resistant transgenic plants, there are concerns that pest populations will develop resistance to the insecticidal proteins produced by these plants. Several strategies have been proposed to protect the effectiveness of Bt-based insect resistance traits, including deploying high doses of protein in combination with refuges, and rotating or co-deploying different toxins (McGaughey et al. (1998), "Bt Resistance Management," Nature Biotechnol. 16: 144-146).

[0013] Insect resistance to Bt proteins can occur through several mechanisms (Heckel et al., 2007, Piggot and Ellar, 2007). Multiple Cry protein receptor protein classes have been identified in insects, and each receptor class has multiple examples. Resistance to specific Cry proteins can occur through mutations within the toxin-binding portion of the cadherin domain of the receptor protein, for example. Another form of resistance can be mediated by protoxin-processing proteases. Therefore, resistance to Cry1A toxins in Lepidoptera species has a complex genetic basis, with at least four different major resistance genes. Resistance to Cry proteins has already emerged in the field in Plutella xylostella (Tabashnik, 1994), Trichoplusia ni (Janmaat and Myers 2003, 2005), Helicoverpa zea (Tabashnik et al., 2008), and Spodoptera frugiperda (Storer et al., 2010), among other Lepidoptera species. Developing new, highly potent Cry proteins will provide more tools for managing Lepidoptera.

[0014] The present invention provides Bt insecticidal proteins that are effective in controlling insects resistant to Cry1Ac and Cry1F. These protein toxins can be advantageously used to protect agronomic crops from insect feeding damage. The ability to express these insect toxins in a manner that results in sufficient amounts of functionally active protein in the crop of interest is also a subject of the present invention. SUMMARY OF THE INVENTION

[0015] A modified CrylCa toxin comprising residues 2 to 68 of SEQ ID NO: 2, wherein amino acid residue 54 is selected from Gly and Ala, amino acid residue 57 is selected from Leu and Met, and amino acid residue 68 is selected from Val, Phe, and Ile. A modified CrylCa toxin comprising residues 2 to 628 of SEQ ID NO: 210, wherein amino acid residue 54 is selected from Gly and Ala, amino acid residue 57 is selected from Leu and Met, amino acid residue 68 is selected from Val, Phe, and Ile, amino acid residue 73 is selected from Trp, Ala, and Met, amino acid residue 596 is selected from Phe, Met, and Ala, and amino acid residue 620 is selected from Leu and Phe. The modified CrylCa toxin further comprises a carboxyl-terminal extension consisting of amino acid residues 629 to 1164 of SEQ ID NO: 36. The modified CrylCa toxin further comprises a carboxyl-terminal extension consisting of amino acid residues 629 to 1164 of SEQ ID NO: 36. The modified Cry1Ca toxin further comprises an amino-terminal extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40. The modified Cry1Ca toxin further comprises an amino-terminal extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40. The modified Cry1Ca toxin further comprises an amino-terminal extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40. The modified Cry1Ca toxin further comprises an amino-terminal extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40.

[0016] The present invention includes DNA encoding a modified Cry1Ca toxin, a transgenic plant producing a modified Cry1Ca toxin, and a method of controlling pests using the modified Cry1Ca toxin.

[0017] The present invention relates to novel materials and methods for controlling arthropod pests that are harmful to plants and agriculture.In preferred embodiments, the present invention provides materials and methods for controlling lepidopteran pests.

[0018] Specific BtCry proteins (endotoxins, toxins) useful according to the present invention include toxins obtainable from the Bt isolate designated MR-1206. The present invention also encompasses the use of mutants of the exemplary Bt isolates and toxins that have improved lepidopteran activity, are resistant to protease processing, or are expressed at high levels when the gene is transformed into a heterologous expression system. Procedures for preparing mutants are well known in the art of microbiology. Ultraviolet light and chemical mutagens such as nitrosoguanidine are widely used for this purpose.

[0019] The subject protein toxin can be "applied" or provided to contact the target insect in a variety of ways. For example, transgenic plants (wherein the protein is produced by and present in the plant) can be used, which are well known in the art. The toxin gene can also be selectively expressed in specific tissues of the plant, such as roots, leaves, etc. This can be achieved by using, for example, tissue-specific promoters. Spray-on application is another example and is also known in the art. The subject protein can be appropriately formulated for the desired end use and then sprayed (or otherwise applied) onto and / or around and / or near the plant to be protected before an infestation is detected, after an infestation is detected, after the target insect is detected, before and after, etc. The subject protein can also be appropriately formulated and applied to seeds in the form of a seed treatment, which allows the protein to contact the root zone of the plant to protect it from root-eating insects. For example, bait granules can also be used and are known in the art.

[0020] The proteins of the present invention can be used to protect nearly any type of plant from damage by Lepidoptera. Examples of such plants include corn, sunflower, soybean, cotton, canola, rice, sorghum, wheat, barley, vegetables, ornamentals, peppers (including peppers), sugar beets, fruit, and turfgrass, to name a few. Particularly preferred plants are corn, soybean, and cotton. A most preferred plant is corn. Another most preferred plant is soybean. Another most preferred plant is cotton.

[0021] In one embodiment of the present invention, the polynucleotide sequences of the present invention encode toxins of approximately 68-71 kDa. These toxins are used to control lepidopteran pests, particularly fall armyworm, diamondback moth, southwestern corn borer, southern armyworm, corn ear moth, and European corn borer. In a preferred embodiment, the subject invention relates to plant cells transformed with at least one polynucleotide sequence of the subject invention, such that the transformed plant cells produce and contain the insecticidal toxins of the present invention in tissues consumed by the target pest.

[0022] Alternatively, the Bt isolates of the present invention, or recombinant microorganisms expressing genes encoding the insecticidal toxin proteins described herein, can be used to control insect pests. In this regard, the present invention encompasses treating substantially intact Bt cells, and / or recombinant cells containing the toxins of the present invention, such that these cells are treated to prolong insecticidal activity when the substantially intact cells are applied to the environment of a target pest. The treated cells act as a protective coating for the insecticidal toxin. Once ingested by the target insect, the toxin becomes active.

[0023] One aspect of the present invention relates to isolated nucleic acid molecules comprising nucleotide sequences encoding insecticidal proteins and polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acids encoding the claimed toxins. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA), as well as analogs of DNA or RNA generated using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0024] The nucleotide sequences encoding the proteins of the present invention include SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and their complements. "Complement" refers to a nucleotide sequence that has sufficient complementarity with a given nucleotide sequence so that it can hybridize with the given nucleotide sequence to form a stable duplex (double-stranded) molecule. The corresponding amino acid sequences of the insecticidal modified CrylCa toxins encoded by these nucleotide sequences are listed in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

[0025] Nucleic acid molecules that are fragments of the claimed nucleotide sequences encoding the toxins are also encompassed by the present invention. A "fragment" refers to a portion of a nucleotide sequence encoding a claimed modified CrylCa toxin fragment. A fragment of a nucleotide sequence may encode a biologically active portion of the claimed toxin protein, or may be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed below.

[0026] Brief Description of Sequence Listing

[0027] SEQ ID NO: 1 DNA sequence encoding DIG-468

[0028] SEQ ID NO: 2 is the DIG-468 protein sequence

[0029] SEQ ID NO: 3 DNA sequence encoding DIG-483

[0030] SEQ ID NO: 4 is the DIG-483 protein sequence

[0031] SEQ ID NO:5 DNA sequence encoding DIG-485

[0032] SEQ ID NO: 6 is the DIG-485 protein sequence

[0033] SEQ ID NO:7 DNA sequence encoding DIG-487

[0034] SEQ ID NO: 8 is the DIG-487 protein sequence

[0035] SEQ ID NO: 9 DNA sequence encoding DIG-462

[0036] SEQ ID NO: 10 is the DIG-462 protein sequence

[0037] SEQ ID NO: 11 DNA sequence encoding DIG-463

[0038] SEQ ID NO: 12 is the DIG-463 protein sequence

[0039] SEQ ID NO: 13 DNA sequence encoding DIG-464

[0040] SEQ ID NO: 14 is the DIG-464 protein sequence

[0041] SEQ ID NO: 15 DNA sequence encoding DIG-465

[0042] SEQ ID NO: 16 is the DIG-465 protein sequence

[0043] SEQ ID NO: 17 DNA sequence encoding DIG-466

[0044] SEQ ID NO: 18 is the DIG-466 protein sequence

[0045] SEQ ID NO: 19 DNA sequence encoding DIG-467

[0046] SEQ ID NO: 20 is the DIG-467 protein sequence

[0047] SEQ ID NO: 21 DNA sequence encoding DIG-469

[0048] SEQ ID NO: 22 is the DIG-469 protein sequence

[0049] SEQ ID NO: 23 DNA sequence encoding DIG-473

[0050] SEQ ID NO: 24 is the DIG-473 protein sequence

[0051] SEQ ID NO: 25 DNA sequence encoding DIG-474

[0052] SEQ ID NO: 26 is the DIG-474 protein sequence

[0053] SEQ ID NO: 27 DNA sequence encoding DIG-482

[0054] SEQ ID NO: 28 is the DIG-482 protein sequence

[0055] SEQ ID NO: 29 DNA sequence encoding modified Cry1Ca after maize codon optimization (IRDIG544.11)

[0056] SEQ ID NO: 30 is a modified Cry1Ca protein toxin sequence (IRDIG544.11)

[0057] SEQ ID NO: 31 DNA sequence encoding a modified Cry1Ca, IRDIG544.12, which has been optimized for high GC codons

[0058] SEQ ID NO:32 Protein toxin sequence of IRDIG544.12

[0059] SEQ ID NO: 33 is a DNA sequence encoding modified Cry1Ca: IRDIG544.9 optimized for dicotyledons

[0060] SEQ ID NO: 34 Modified Cry1Ca: IRDIG544.9 protein sequence

[0061] SEQ ID NO: 35 Dicotyledonous plant optimized DNA sequence encoding modified Cry1Ca: IRDIG544.8

[0062] SEQ ID NO: 36 IRDIG544.8 protein codon-optimized for dicots

[0063] SEQ ID NO: 37 DNA sequence encoding the fusion of the Cry1Ab protoxin segment and the modified Cry1Ca toxin

[0064] SEQ ID NO:38 is the protein toxin sequence produced from the DNA of SEQ ID NO:37

[0065] SEQ ID NO:39 High GC codon optimized DNA sequence encoding a fusion of modified Cry1Ca IRDIG544.12 and Trap12

[0066] SEQ ID NO:40 is a fusion of the modified CrylCa toxin IRDIG544.12 and TraP12. Sequence Listing <110> Dow AgroSciences <120> Modified Cry1Ca toxins useful for controlling insect pests <130> 70951 <160> 40 <170> PatentIn version 3.5 <210> 1 <211> 1878 <212> DNA <213> Artificial sequence <220> <223> Synthetic coding region <400> 1 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg gtggcttcct tgttggcctt 180 atcgacttcg tctggggaat tgtccagtgg gatgcgtttc tggtgcagat agagcagctg 240 atcaacgaga ggatcgctga gttcgcgaga aatgctgcaa tcgccaacct tgaagggctt 300 ggcaacaact tcaacatcta cgtggaggcg ttcaaggagt gggaagagga ccctaagaat 360 ccagcgacca gaacgagggt tatagatcgg ttccgcatcc tcgatggcct tttggagagg 420 gacatcccga gcttccgcat ttcgggattt gaggttcctc tgctctcagt ctacgctcaa 480 gctgctaatc tgcatctggc catcttgagg gattcagtca tctttggcga acgctggggt 540 cttacgacta tcaacgtgaa cgagaactac aatcggttga ttcggcacat agacgagtat 600 gccgaccact gtgctaacac ctacaatagg ggtctgaaca atctgccaaa gtcaacgtat 660 caagactgga taacctacaa taggctcaga cgggacctca ctctcaccgt gctggacata 720 gctgccttct ttccgaacta cgacaaccgg agatatccta ttcaacccgt tggtcagctc 780 actcgcgagg tctacaccga tccctcatc aacttcaatc cccagctgca atcggtcgca 840 cagctgccca ccttcaatgt gatggaaaac tcagcgatcc ggaatcccca tctgtttgac 900 atacttaaca acctcactat cttcaccgat tggttttcag ttggacgcaa cttctactgg 960 ggagggcaca gagtgatttc aagcctcatt ggaggaggga acattacatc gcctatctat 1020 ggaagggagg ccaaccaaga gccaccaagg tctttcacct tcaacggtcc ggtgttcaga 1080 acacttagca atcccacatt gcgcttgctg caacagccgt ggccagcacc accattcaat 1140 ctgaggggag tggagggtgt ggagttctcg acgcctacaa actcctttac gtacagaggc 1200 agagggacag tggactcact gacagaactc ccacctgagg acaactctgt tcctccgagg 1260 gagggctact cgcaccggct ttgccatgcc accttcgtcc agaggtctgg cacgcctttt 1320 ctgaccactg gggttgtctt tagctggact caccgctcag cgacgctgac caacacaatc 1380 gacccagaga ggatcaatca gatccctctg gtgaagggct ttcgcgtttg gggtggcaca 1440 agcgtgatca ccggacctgg tttcactggt ggggatatcc tcagacgcaa tacgtttggc 1500 gatttcgtga gccttcaagt caacatcaat tccccaatca cccagagata tcggctccgc 1560 ttcagatacg cctcatccag agacgcaagg gtcatcgtcc ttactggagc agccagcacc 1620 ggagtcggag gccaagttag cgtcaacatg ccgttgcaga aaacgatgga aatcggtgaa 1680 aacctcacca gcagaacctt tcgctataca gatttcagca accctttctc cttcagagcc 1740 aatccggaca taatcggcat atccgagcag cccttgttcg gtgctgggtc catctcttct 1800 ggcgagctgt acatcgacaa gattgagatc attctcgcag atgcgactct ggaggctgaa 1860 tcggatcttg aaaggtga 1878 <210> 2 <211> 625 <212> PRT <213> Artificial sequence <220> <223> Translated from a self-synthesized coding region <400> 2 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 [[ID=​​​​​​​​​​​​​​​​​​​​​​​Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gln 145 150 155 160 Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly 165 170 175 Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg 180 185 190 Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr 195 200 205 Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp Ile 210 215 220 Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile 225 230 235 240 Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro 245 250 255 Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe 260 265 270 Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met 275 280 285 Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn 290 295 300 Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp 305 310 315 320 Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr 325 330 335 Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe 340 345 350 Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg 355 360 365 Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val 370 375 380 Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly 385 390 395 400 Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser 405 410 415 Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe 420 425 430 Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser 435 440 445 Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg 450 455 460 Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr 465 470 475 480 Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg 485 490 495 Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro 500 505 510 Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp 515 520 525 Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly 530 535 540 Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu 545 550 555 560 Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe 565 570 575 Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu 580 585 590 Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile 595 600 605 Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser Asp Leu Glu 610 615 620 Arg 625 <210> 3 <211> 1878 <212> DNA <213> Artificial sequence <220> <223> Synthetic coding region <400> 3 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg ccggcttcct tgttggcctt 180 atcgacttcg tctggggaat tgtccagtgg gatgcgtttc tggtgcagat agagcagctg 240 [[ID=2⑨]]atcaacgaga ggatcgctga gttcgcgaga aatgctgcaa tcgccaacct tgaagggctt 300 ggcaacaact tcaacatcta cgtggaggcg ttcaaggagt gggaagagga ccctaagaat 360 ccagcgacca gaacgagggt tatagatcgg ttccgcatcc tcgatggcct tttggagagg 420 gacatcccga gcttccgcat ttcgggattt gaggttcctc tgctctcagt ctacgctcaa 480 gctgctaatc tgcatctggc catcttgagg gattcagtca tctttggcga acgctggggt 540 cttacgacta tcaacgtgaa cgagaactac aatcggttga ttcggcacat agacgagtat 600 gccgaccact gtgctaacac ctacaatagg ggtctgaaca atctgccaaa gtcaacgtat 660 caagactgga taacctacaa taggctcaga cgggacctca ctctcaccgt gctggacata 720 gctgccttct ttccgaacta cgacaaccgg agatatccta ttcaacccgt tggtcagctc 780 actcgcgagg tctacaccga tccctcatc aacttcaatc cccagctgca atcggtcgca 840 cagctgccca ccttcaatgt gatggaaaac tcagcgatcc ggaatcccca tctgtttgac 900 atacttaaca acctcactat cttcaccgat tggttttcag ttggacgcaa cttctactgg 960 ggagggcaca gagtgatttc aagcctcatt ggaggaggga acattacatc gcctatctat 1020 ggaagggagg ccaaccaaga gccaccaagg tctttcacct tcaacggtcc ggtgttcaga 1080 acacttagca atcccacatt gcgcttgctg caacagccgt ggccagcacc accattcaat 1140 ctgaggggag tggagggtgt ggagttctcg acgcctacaa actcctttac gtacagaggc 1200 agagggacag tggactcact gacagaactc ccacctgagg acaactctgt tcctccgagg 1260 gagggctact cgcaccggct ttgccatgcc accttcgtcc agaggtctgg cacgcctttt 1320 ctgaccactg gggttgtctt tagctggact caccgctcag cgacgctgac caacacaatc 1380 gacccagaga ggatcaatca gatccctctg gtgaagggct ttcgcgtttg gggtggcaca 1440 agcgtgatca ccggacctgg tttcactggt ggggatatcc tcagacgcaa tacgtttggc 1500 gatttcgtga gccttcaagt caacatcaat tccccaatca cccagagata tcggctccgc 1560 ttcagatacg cctcatccag agacgcaagg gtcatcgtcc ttactggagc agccagcacc 1620 ggagtcggag gccaagttag cgtcaacatg ccgttgcaga aaacgatgga aatcggtgaa 1?80 aacctcacca gcagaacctt tcgctataca gatttcagca accctttctc cttcagagcc 1740 aatccggaca taatcggcat atccgagcag cccttgttcg gtgctgggtc catctcttct 1800 ggcgagctgt acatcgacaa gattgagatc attctcgcag atgcgactct ggaggctgaa 1860 tcggatcttg aaaggtga 1878 [[ID=?1]] <210> 4 <211> 625 <212> PRT<??00347><213> Artificial Sequence <220> <223> Translated from a synthetic coding region It should be noted that there seems to be an error in "1?80" in line 12, which is likely a typo. It should probably be "1680".<400> 4 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Ala Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu 65 70 75 80 Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn 85 90 95 Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys 100 105 110 Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr Arg Val Ile 115 120 125 Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser 130 135 140 Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gln 145 150 155 160 Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly 165 170 175 Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg 180 185 190 Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr 195 200 205 Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp Ile 210 215 220 Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile 225 230 235 240 Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro 245 250 255 Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe 260 265 270 Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met 275 280 285 Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn 290 295 300 Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp 305 310 315 320 Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr 325 330 335 Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe 340 345 350 Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg 355 360 365 Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val 370 375 380 Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly 385 390 395 400 Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser 405 410 415 Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe 420 425 430 Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser 435 440 445 Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg 450 455 460 Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr 465 470 475 480 Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg 485 490 495 Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro 500 505 510 Island Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp 515 520 525 Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly 530 535 540 Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu 545 550 555 560 Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe 565 570 575 Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu 580 585 590 Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile 595 600 605 Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser Asp Leu Glu 610 615 620 Arg 625 <210> 5 <211> 1878 <212> DNA <213> Artificial sequence <220> <223> Synthetic coding region <400> 5 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg gtggcttcgc cgttggcctt 180 atcgacttcg tctggggaat tgtccagtgg gatgcgtttc tggtgcagat agagcagctg 240 atcaacgaga ggatcgctga gttcgcgaga aatgctgcaa tcgccaacct tgaagggctt 300 ggcaacaact tcaacatcta cgtggaggcg ttcaaggagt gggaagagga ccctaagaat 360 ccagcgacca gaacgagggt tatagatcgg ttccgcatcc tcgatggcct tttggagagg 420 gacatcccga gcttccgcat ttcgggattt gaggttcctc tgctctcagt ctacgctcaa 480 gctgctaatc tgcatctggc catcttgagg gattcagtca tctttggcga acgctggggt 540 cttacgacta tcaacgtgaa cgagaactac aatcggttga ttcggcacat agacgagtat 600 gccgaccact gtgctaacac ctacaatagg ggtctgaaca atctgccaaa gtcaacgtat 660 caagactgga taacctacaa taggctcaga cgggacctca ctctcaccgt gctggacata 720 gctgccttct ttccgaacta cgacaaccgg agatatccta ttcaacccgt tggtcagctc 780 actcgcgagg tctacaccga tccctcatc aacttcaatc cccagctgca atcggtcgca 840 cagctgccca ccttcaatgt gatggaaaac tcagcgatcc ggaatcccca tctgtttgac 900 atacttaaca acctcactat cttcaccgat tggttttcag ttggacgcaa cttctactgg 960 ggagggcaca gagtgatttc aagcctcatt ggaggaggga acattacatc gcctatctat 1020 ggaagggagg ccaaccaaga gccaccaagg tctttcacct tcaacggtcc ggtgttcaga 1080 acacttagca atcccacatt gcgcttgctg caacagccgt ggccagcacc accattcaat 1140 ctgaggggag tggagggtgt ggagttctcg acgcctacaa actcctttac gtacagaggc 1200 agagggacag tggactcact gacagaactc ccacctgagg acaactctgt tcctccgagg 1260 gaggctact cgcaccggct ttgccatgcc accttcgtcc agaggtctgg cacgcctttt 1320 ctgaccactg gggttgtctt tagctggact caccgctcag cgacgctgac caacacaatc 1380 gacccagaga ggatcaatca gatccctctg gtgaagggct ttcgcgtttg gggtggcaca 1440 agcgtgatca ccggacctgg tttcactggt ggggatatcc tcagacgcaa tacgtttggc 1500 gatttcgtga gccttcaagt caacatcaat tccccaatca cccagagata tcggctccgc 1560 ttcagatacg cctcatccag agacgcaagg gtcatcgtcc ttactggagc agccagcacc 1620 ggagtcggag gccaagttag cgtcaacatg ccgttgcaga aaacgatgga aatcggtgaa 1680 aacctcacca gcagaacctt tcgctataca gatttcagca accctttctc cttcagagcc 1740 aatccggaca taatcggcat atccgagcag cccttgttcg gtgctgggtc catctcttct 1800 ggcgagctgt acatcgacaa gattgagatc attctcgcag atgcgactct ggaggctgaa 1860 tcggatcttg aaaggtga 1878 <210> 6 <211> 625 <212> PRT <213> Artificial Sequence <220> <223> Translated from a synthetic coding region <400> 6 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Ala Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu 65 70 75 80 Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn 85 90 95 Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys 100 105 110 Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr Arg Val Ile 115 120 125 Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser 130 135 140 Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gln 145 150 155 160 Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly 165 170 175 Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg 180 185 190 Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr 195 200 205 Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp Ile 210 215 220 Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile 225 230 235 240 Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro 245 250 255 Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe 260 265 270 Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met 275 280 285 Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn 290 295 300 Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp 305 310 315 320 Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr 325 330 335 Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe 340 345 350 Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg 355 360 365 Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val 370 375 380 Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly 385 390 395 400 Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser 405 410 415 Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe 420 425 430 Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser 435 440 445 Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg 450 455 460 Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr 465 470 475 480 Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg 485 490 495 Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro 500 505 510 Island Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp 515 520 525 Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly 530 535 540 Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu 545 550 555 560 Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe 565 570 575 Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu 580 585 590 Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile 595 600 605 Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser Asp Leu Glu 610 615 620 Arg 625 <210> 7 <211> 1878 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Coding Region <400> 7 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg gtggcttcat ggttggcctt 180 atcgacttcg tctggggaat tgtccagtgg gatgcgtttc tggtgcagat agagcagctg 240 atcaacgaga ggatcgctga gttcgcgaga aatgctgcaa tcgccaacct tgaagggctt 300 ggcaacaact tcaacatcta cgtggaggcg ttcaaggagt gggaagagga ccctaagaat 360 ccagcgacca gaacgagggt tatagatcgg ttccgcatcc tcgatggcct tttggagagg 420 gacatcccga gcttccgcat ttcgggattt gaggttcctc tgctctcagt ctacgctcaa 480 gctgctaatc tgcatctggc catcttgagg gattcagtca tctttggcga acgctggggt 540 cttacgacta tcaacgtgaa cgagaactac aatcggttga ttcggcacat agacgagtat 600 gccgaccact gtgctaacac ctacaatagg ggtctgaaca atctgccaaa gtcaacgtat 660 caagactgga taacctacaa taggctcaga cgggacctca ctctcaccgt gctggacata 720 gctgccttct ttccgaacta cgacaaccgg agatatccta ttcaacccgt tggtcagctc 780 actcgcgagg tctacaccga tccctcatc aacttcaatc cccagctgca atcggtcgca 840 cagctgccca ccttcaatgt gatggaaaac tcagcgatcc ggaatcccca tctgtttgac 900 atacttaaca acctcactat cttcaccgat tggttttcag ttggacgcaa cttctactgg 960 ggagggcaca gagtgatttc aagcctcatt ggaggaggga acattacatc gcctatctat 1020 ggaagggagg ccaaccaaga gccaccaagg tctttcacct tcaacggtcc ggtgttcaga 1080 acacttagca atcccacatt gcgcttgctg caacagccgt ggccagcacc accattcaat 1140 ctgaggggag tggagggtgt ggagttctcg acgcctacaa actcctttac gtacagaggc 1200 agagggacag tggactcact gacagaactc ccacctgagg acaactctgt tcctccgagg 1260 gaggctact cgcaccggct ttgccatgcc accttcgtcc agaggtctgg cacgcctttt 1320 ctgaccactg gggttgtctt tagctggact caccgctcag cgacgctgac caacacaatc 1380 gacccagaga ggatcaatca gatccctctg gtgaagggct ttcgcgtttg gggtggcaca 1440 agcgtgatca ccggacctgg tttcactggt ggggatatcc tcagacgcaa tacgtttggc 1500 gatttcgtga gccttcaagt caacatcaat tccccaatca cccagagata tcggctccgc 1560 ttcagatacg cctcatccag agacgcaagg gtcatcgtcc ttactggagc agccagcacc 1620 ggagtcggag gccaagttag cgtcaacatg ccgttgcaga aaacgatgga aatcggtgaa 1680 aacctcacca gcagaacctt tcgctataca gatttcagca accctttctc cttcagagcc 1740 aatccggaca taatcggcat atccgagcag cccttgttcg gtgctgggtc catctcttct 1800 ggcgagctgt acatcgacaa gattgagatc attctcgcag atgcgactct ggaggctgaa 1860 tcggatcttg aaaggtga 1878 <210> 8 <211> 625 <212> PRT <213> Artificial Sequence <220> <223> Translated from a synthetic coding region <400> 8 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Met Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu 65 70 75 80 Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn 85 90 95 Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys 100 105 110 Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr Arg Val Ile 115 120 125 Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser 130 135 140 Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gln 145 150 155 160 Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly 165 170 175 Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg 180 185 190 Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr 195 200 205 Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp Ile 210 215 220 Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile 225 230 235 240 Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro 245 250 255 Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe 260 265 270 Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met 275 280 285 Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn 290 295 300 Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp 305 310 315 320 Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr 325 330 335 Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe 340 345 350 Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg 355 360 365 Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val 370 375 380 Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly 385 390 395 400 Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser 405 410 415 Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe 420 425 430 Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser 435 440 445 Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg 450 455 460 Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr 465 470 475 480 Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg 485 490 495 Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro 500 505 510 Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp 515 520 525 Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly 530 535 540 Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu 545 550 555 560 Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe 565 570 575 Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu 580 585 590 Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile 595 600 605 Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser Asp Leu Glu 610 615 620 Arg 625 <210> 9 <211> 1887 <212> DNA <213> Bacillus thuringiensis <400> 9 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg gtggcttcct tgttggcctt 180 atcgacttcg tctggggaat tgtcggaccc tcccagtggg atgcgtttct ggtgcagata 240 gagcagctga tcaacgagag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaggcgt tcaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaac gagaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gtctgaacaa tctgccaaag 660 tcaacgtatc aagactggat aacctacaat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat cccctcatca acttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctgtttgaca tacttaacaa cctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgatttca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaagag ccaccaaggt ctttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 ccattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggctt tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aacacaatcg acccagagag gatcaatcag atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacatcaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcaacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaaccttt cgctatacag atttcagcaa ccctttctcc 1740 ttcagagcca atccggacat aatcggcata tccgagcagc ccttgttcgg tgctgggtcc 1800 atctcttctg gcgagctgta catcgacaag attgagatca ttctcgcaga tgcgactctg 1860 gaggctgaat cggatcttga aaggtga 1887 <210> 10 <211> 628 <212> PRT <213> Bacillus thuringiensis <400> 10 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 11 <211> 1887 <212> DNA <213> artificial sequence <220> <223> synthetic coding district <400> 11 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg ccggcttcct tgttggcctt 180 atcgacttcg tctggggaat tgtcggaccc tcccagtggg atgcgtttct ggtgcagata 240 gagcagctga tcaacgagag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaggcgt tcaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaac gagaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gtctgaacaa tctgccaaag 660 tcaacgtatc aagactggat aacctacaat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat cccctcatca acttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctgtttgaca tacttaacaa cctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgatttca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaagag ccaccaaggt ctttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 ccattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggctt tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aacacaatcg acccagagag gatcaatcag atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacatcaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcaacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaaccttt cgctatacag atttcagcaa ccctttctcc 1740 ttcagagcca atccggacat aatcggcata tccgagcagc ccttgttcgg tgctgggtcc 1800 atctcttctg gcgagctgta catcgacaag attgagatca ttctcgcaga tgcgactctg 1860 gaggctgaat cggatcttga aaggtga 1887 <210> 12 <211> 628[[ID=1'7]] <212> PRT <><213> Artificial Sequence <220> <223> Translated from a synthetic coding region <400> 12 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 It should be noted that there may be some inaccuracies in the translation due to the complexity and potential ambiguity of the original text, especially in the context of patent - specific jargon. The above translation attempts to be as faithful as possible while maintaining the integrity of the original text structure and tags.Asn Phe Val Pro Gly Ala Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 13 <211> 1887 <212> DNA <213> Artificial sequence <220> <223> Synthetic coding region <400> 13 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg gtggcttcat ggttggcctt 180 atcgacttcg tctggggaat tgtcggaccc tcccagtggg atgcgtttct ggtgcagata 240 gagcagctga tcaacgagag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaggcgt tcaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaac gagaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gtctgaacaa tctgccaaag 660 tcaacgtatc aagactggat aacctacaat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat cccctcatca acttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctgtttgaca tacttaacaa cctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgatttca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaagag ccaccaaggt ctttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 ccattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggctt tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aacacaatcg acccagagag gatcaatcag atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacatcaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcaacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaaccttt cgctatacag atttcagcaa ccctttctcc 1740 ttcagagcca atccggacat aatcggcata tccgagcagc ccttgttcgg tgctgggtcc 1800 atctcttctg gcgagctgta catcgacaag attgagatca ttctcgcaga tgcgactctg 1860 gaggctgaat cggatcttga aaggtga 1887 <210> 14 <211> 628 <212> PRT <213> Artificial Sequence <220> <223> Translated from a synthetic coding region <400> 14 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Met Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210>​​​​​​​​​​​​​​​​​​​atcgacttcg tctggggaat tgtcggaccc tcccagtggg atgcgtttct ggtgcagata 240 gagcagctga tcaacgagag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaggcgt tcaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaac gagaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gtctgaacaa tctgccaaag 660 tcaacgtatc aagactggat aacctacaat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat cccctcatca acttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctgtttgaca tacttaacaa cctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgatttca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaagag ccaccaaggt ctttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 ccattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggctt tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aacacaatcg acccagagag gatcaatcag atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacatcaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcaacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaaccttt cgctatacag atttcagcaa ccctttctcc 1740 ttcagagcca atccggacat aatcggcata tccgagcagc ccttgttcgg tgctgggtcc 1800 atctcttctg gcgagctgta catcgacaag attgagatca ttctcgcaga tgcgactctg 1860 gaggctgaat cggatcttga aaggtga 1887 <210> 16 <211> 628 <212> PRT <213> Artificial Sequence <220> <223> Translated from a synthetic coding region <400> 16 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Ala Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 17 <211> 1887 <212> DNA <213> Synthetic sequence <220> <223> Synthetic coding region<​​​​​​​​atcgacttcg tctggggaat tttcggaccc tcccagtggg atgcgtttct ggtgcagata 240 gagcagctga tcaacgagag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaggcgt tcaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaac gagaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gtctgaacaa tctgccaaag 660 tcaacgtatc aagactggat aacctacaat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat cccctcatca acttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctgtttgaca tacttaacaa cctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgatttca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaagag ccaccaaggt ctttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 ccattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggctt tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aacacaatcg acccagagag gatcaatcag atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacatcaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcaacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaaccttt cgctatacag atttcagcaa ccctttctcc 1740 ttcagagcca atccggacat aatcggcata tccgagcagc ccttgttcgg tgctgggtcc 1800 atctcttctg gcgagctgta catcgacaag attgagatca ttctcgcaga tgcgactctg 1860 gaggctgaat cggatcttga aaggtga 1887 <210> 18 <211> 628 <212> PRT <213> Artificial Sequence <220> <223> Translated from a synthetic coding region <400> 18 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 19 <211> 1887 <212> DNA <213> Artificial sequence <220> <223> Synthetic coding region <400> 19 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg gtggcttcct tgttggcctt 180 atcgacttcg tctggggaat tatcggaccc tcccagtggg atgcgtttct ggtgcagata 240 gagcagctga tcaacgagag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaggcgt tcaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaac gagaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gtctgaacaa tctgccaaag 660 tcaacgtatc aagactggat aacctacaat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat cccctcatca acttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctgtttgaca tacttaacaa cctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgatttca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaagag ccaccaaggt ctttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 ccattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggctt tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aacacaatcg acccagagag gatcaatcag atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacatcaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcaacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaaccttt cgctatacag atttcagcaa ccctttctcc 1740 ttcagagcca atccggacat aatcggcata tccgagcagc ccttgttcgg tgctgggtcc 1800 atctcttctg gcgagctgta catcgacaag attgagatca ttctcgcaga tgcgactctg 1860 gaggctgaat cggatcttga aaggtga 1887 <210> 20 <211> 628 <212> PRT<00013​​​​​​​​​​​​​​​​​​​​​​​​​​65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 21 <211> 1887 <212> DNA <213> Artificial sequence <220> <223> Synthetic coding region <400> 21 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg gtggcttcct tgttggcctt 180 atcgacttcg tctggggaat tgtcggaccc tcccaggcgg atgcgtttct ggtgcagata 240 gagcagctga tcaacgagag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaggcgt tcaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaac gagaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gtctgaacaa tctgccaaag 660 tcaacgtatc aagactggat aacctacaat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat cccctcatca acttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctgtttgaca tacttaacaa cctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgatttca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaagag ccaccaaggt ctttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 ccattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggctt tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aacacaatcg acccagagag gatcaatcag atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacatcaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcaacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaaccttt cgctatacag atttcagcaa ccctttctcc 1740 ttcagagcca atccggacat aatcggcata tccgagcagc ccttgttcgg tgctgggtcc 1800 atctcttctg gcgagctgta catcgacaag attgagatca ttctcgcaga tgcgactctg 1860 gaggctgaat cggatcttga aaggtga 1887 <210> 22 <211> 628 <212> PRT <213> Artificial Sequence <220> <223> Translated from a synthetic coding region <400> 22 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Ala Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 23 <211> 1887 <212> DNA <213> Artificial sequence <220> <223> Synthetic coding region <400> 23 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg gtggcttcct tgttggcctt 180 atcgacttcg tctggggaat tgtcggaccc tcccagtggg atgcgtttct ggtgcagata 240 gagcagctga tcaacgagag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaggcgt tcaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaac gagaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gtctgaacaa tctgccaaag 660 tcaacgtatc aagactggat aacctacaat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat cccctcatca acttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctgtttgaca tacttaacaa cctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgatttca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaagag ccaccaaggt ctttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 ccattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggctt tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aacacaatcg acccagagag gatcaatcag atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacatcaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcaacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaaccttt cgctatacag atttcagcaa ccctttctcc 1740 ttcagagcca atccggacat aatcggcata tccgagcagc ccttgatggg tgctgggtcc 1800 atctcttctg gcgagctgta catcgacaag attgagatca ttctcgcaga tgcgactctg 1860 gaggctgaat cggatcttga aaggtga 1887 <210> 24 <211> 628 <212> PRT <213> Artificial Sequence <220> <223> Translated from a synthetic coding region <400> 24 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Met Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 25 <211> 1887 <212> DNA <213> artificial sequence <220> <223> Synthesized coding district <400> 25 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg gtggcttcct tgttggcctt 180 atcgacttcg tctggggaat tgtcggaccc tcccagtggg atgcgtttct ggtgcagata 240 gagcagctga tcaacgagag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaggcgt tcaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaac gagaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gtctgaacaa tctgccaaag 660 tcaacgtatc aagactggat aacctacaat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat cccctcatca acttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctgtttgaca tacttaacaa cctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgatttca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaagag ccaccaaggt ctttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 ccattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggctt tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aacacaatcg acccagagag gatcaatcag atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacatcaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcaacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaaccttt cgctatacag atttcagcaa ccctttctcc 1740 ttcagagcca atccggacat aatcggcata tccgagcagc ccttggccgg tgctgggtcc 1800 atctcttctg gcgagctgta catcgacaag attgagatca ttctcgcaga tgcgactctg 1860 gaggctgaat cggatcttga aaggtga 1887 <210> 26 <211> 628 <212> PRT <213> Artificial Sequence <220> <223> Translated from a synthetic coding region <400> 26 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Ala Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 27 <211> 1887 <212> DNA <213> Artificial sequence <220> <223> Synthetic coding region <400> 27 atggataaca accccaacat taacgagtgc atcccgtaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccatcga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg ccggcttcct tgttggcctt 180 atcgacttcg tctggggaat tgtcggaccc tcccagatgg atgcgtttct ggtgcagata 240 gagcagctga tcaacgagag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaggcgt tcaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaac gagaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gtctgaacaa tctgccaaag 660 tcaacgtatc aagactggat aacctacaat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat cccctcatca acttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctgtttgaca tacttaacaa cctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgatttca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaagag ccaccaaggt ctttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 ccattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggctt tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aacacaatcg acccagagag gatcaatcag atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacatcaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcaacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaaccttt cgctatacag atttcagcaa ccctttctcc 1740 ttcagagcca atccggacat aatcggcata tccgagcagc ccttgttcgg tgctgggtcc 1800 atctcttctg gcgagctgta catcgacaag attgagatca ttctcgcaga tgcgactctg 1860 gaggctgaat cggatcttga aaggtga 1887 <210> 28 <211> 628 <212> PRT <213> Artificial Sequence <220> <223> Translated from a synthetic coding region <400> 28 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15<​​​​​​​​​​​​​​65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 29 <211> 1887 <212> DNA <213> artificial sequence <220> <223> Synthesized coding district <400> 29 atggataaca accccaacat taacgagtgc atcccctaca actgcctctc gaatccagaa 60 gaagtgctct tggatggcga gaggatttcg actggcaaca gctccataga catttccctc 120 tccttggttc agttccttgt gtctaacttc gtccctggcg gtggcttcct tgttggatta 180 atcgacttcg tctggggaat tgtcggaccc tcccagtggg atgcgtttct ggtgcagata 240 gagcaattaa ttaatgaaag gatcgctgag ttcgcgagaa atgctgcaat cgccaacctt 300 gaagggcttg gcaacaactt caacatctac gtggaagcat ttaaggagtg ggaagaggac 360 cctaagaatc cagcgaccag aacgagggtt atagatcggt tccgcatcct cgatggcctt 420 ttggagaggg acatcccgag cttccgcatt tcgggatttg aggttcctct gctctcagtc 480 tacgctcaag ctgctaatct gcatctggcc atcttgaggg attcagtcat ctttggcgaa 540 cgctggggtc ttacgactat caacgtgaat gaaaactaca atcggttgat tcggcacata 600 gacgagtatg ccgaccactg tgctaacacc tacaataggg gattaaataa tctgccaaag 660 tcaacgtatc aagactggat aacatataat aggctcagac gggacctcac tctcaccgtg 720 ctggacatag ctgccttctt tccgaactac gacaaccgga gatatcctat tcaacccgtt 780 ggtcagctca ctcgcgaggt ctacaccgat ccattaatta atttcaatcc ccagctgcaa 840 tcggtcgcac agctgcccac cttcaatgtg atggaaaact cagcgatccg gaatccccat 900 ctctttgaca tacttaataa tctcactatc ttcaccgatt ggttttcagt tggacgcaac 960 ttctactggg gagggcacag agtgattca agcctcattg gaggagggaa cattacatcg 1020 cctatctatg gaagggaggc caaccaggag ccaccaaggt cttcacctt caacggtccg 1080 gtgttcagaa cacttagcaa tcccacattg cgcttgctgc aacagccgtg gccagcacca 1140 cattcaatc tgaggggagt ggagggtgtg gagttctcga cgcctacaaa ctcctttacg 1200 tacagaggca gagggacagt ggactcactg acagaactcc cacctgagga caactctgtt 1260 cctccgaggg agggctactc gcaccggct tgccatgcca ccttcgtcca gaggtctggc 1320 acgccttttc tgaccactgg ggttgtcttt agctggactc accgctcagc gacgctgacc 1380 aatacaatcg acccagagag aattaatcaa atccctctgg tgaagggctt tcgcgtttgg 1440 ggtggcacaa gcgtgatcac cggacctggt ttcactggtg gggatatcct cagacgcaat 1500 acgtttggcg atttcgtgag ccttcaagtc aacattaatt ccccaatcac ccagagatat 1560 cggctccgct tcagatacgc ctcatccaga gacgcaaggg tcatcgtcct tactggagca 1620 gccagcaccg gagtcggagg ccaagttagc gtcacatgc cgttgcagaa aacgatggaa 1680 atcggtgaaa acctcaccag cagaacctt cgctatacag atttcagca ccctttctcc 1740 ttcagagcca atccggatat aatcggcata tccgagcagc ccttgttcgg tgctgggtcc 1800 atctcttcag gcgagctgta catcgataaa attgagatca ttctcgcaga tgcgactc 1860 Gaggctgaat Cggatcttga Aggtga 1887 <210> 30 <211> 628 <212> PRT <213> Bacillus thuringiensis <400> 30 Met Asp Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Glu Glu Arg And Ser Thr Gly 20 25 30 Asn To Be To Be There Asp To Be To Be Leo To Be Leo Valley Gln Phe To Be Leu Valley 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 31 <211> 1887 <212> DNA <213> artificial sequence <220> <223> Synthesized coding district <400> 31 atggataaca acccgaacat caacgagtgc atcccctaca actgcctgag caaccccgag 60 gaggtgctgc tggacggcga gaggatctca accggcaaca gcagcatcga catcagcctg 120 tccctggtgc agttcctggt gagcaacttc gtgccgggcg gcggcttcct ggtgggatta 180 atcgacttcg tgtggggcat cgtcggcccg tcccagtggg acgcttcct ggttcagatc 240 gagcaattaa ttaatgaag gatgcagag ttcgcgagga acgcggccat cgccaacctg 300 gaggggcctgg gcacaactt cacacactc gtggaagcat ttaggagtg ggaggaggac 360 cccaagaacc cggccacgag gacgaggggtg atcgaccgct tcgcatcct ggacggcctg 420 ctggagagggg acatcccgtc cttcagaatc agcggctcg aggtccccgct gctgtccgtg 480 tacgcgcaag cggccaacct gcacctggcg atcctgaggg actccgtgat attcggcgag 540 aggtggggcc tgaccacat caacgtgaat gaaactaca accggctgat aaggcacatc 600 gacgagtacg ccgaccactg cgccacacc tacaataggg gattaataa tctgcccaag 660 agcacctacc agactggat cacatataac cggctgagga gggacctgac cctgaccgtg 720 ctggacatcg ccgcgttctt cccgaactac gataagc gctaccgat ccagccggtg 780 ggccagctga cccgcgaggt gtacaccgac ccattaatta atttcaaccc gcagctccag 840 tccgtggccc agctgccgac cttcaacgtg atggacaca gcgccatccg gaacccgcac 900 ctgttcgaca tcctgaataa tctgaccatc ttcaccgact ggttctcagt gggccggac 960 ttctactggg gcggccatag ggtgatctcc agcctgatcg gcggcggcaa catcacctcc 1020 ccgatctacg ggagggaggc gaaccaggag ccgccgaggt ccttcacctt caacggcccg 1080 gtgtttagga ccctgtccaa cccgaccctg aggctgctcc agcagccgtg gccggcgccg 1140 ccgttcaacc tgaggggcgt ggagggcgtg gagttcagca ccccgaccaa cagcttcacc 1200 taccgggggga ggggcaccgt ggactcactg accgagctgc cgccggagga caacagcgtg 1260 ccgccgaggg agggctacag ccataggctg tgccacgcca ccttcgtgca gaggagcggc 1320 accccgttct tgacgaccgg cgtggtgttc tcctggaccc accggagcgc gaccctgacc 1380 aatacaatcg acccggagag aattaatcaa atcccgctgg tgaagggctt ccgggtgtgg 1440 ggcggcacct ccgtgatcac cgggccgggc tttaccggcg gcgacatcct gaggaggaac 1500 acgtcggcg acttcgtgag cctccaagtg aacattaata gcccgatcac ccagcgctac 1560 cggctgaggt tccgctacgc gtcaagccgc gacgcgaggg tgatcgtgct gaccggcgcg 1620 gcctcaaccg gcgtgggcgg ccaagtgtcc gtgaacatgc cgctgcaaaa gacgatggag 1680 atcggcgaga acctgaccctc aaggaccttc cgctacaccg acttcagca cccgttcagc 1740 tttagggcca acccggatat aatcggcatc agcgagcagc cgctgttcgg cgccggctcc 1800 atctcaagcg gcgagctgta catcgataaa atcgagatca tcctggcgga cgcgaccttg 1860 Gaggccgagt ccgacctgga gaggtga 1887 <210> 32 <211> 628 <212> PRT <213> Bacillus thuringiensis <400> 32 Met Asp Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Glu Glu Arg And Ser Thr Gly 20 25 30 Asn To Be To Be There Asp To Be To Be Leo To Be Leo Valley Gln Phe To Be Leu Valley 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu With Asn Glu Arg With Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 33 <211> 1887 <212> DNA <213> artificial sequence <220> <223> Synthesized coding district <400> 33 atggacaaca atcccaatat caatgagtgc atcccataca actgcctttc aaatcccgag 60 gaagctcttac tggatggga aggattagc actggcaatt ccagcattga catttccttg 120 agtctcgttc aattccttgt gagcaacttt gtgcctggcg gagggttctt ggttggtctc 180 atagactttg tttgggggaat tgtaggacca tcacagtggg atgccttctt agtccagatt 240 gaacagctta tcaatgagag aatagctgag ttcgcaagaa atgctgcaat cgctaacttg 300 gagggactcg gcaacaactt caacatctac gtggaggctt tcaaagaatg ggaagaagat 360 cctaagaatc cagctactag gactagagtc atcgataggt ttaggattct tgatgggctg 420 ttggagcgtg acattccttc ctttaggatt agtggcttcg aggttcctct cctttctgtc 480 tatgctcaag cagccaatct ccatctcgcc attctccgtg attcagtaat cttcggagaa 540 agatggggtt tgacaacgat caatgtgaat gagaactaca acagattgat cagacacatt 600 gatgagtatg ctgatcattg tgctaacaca tacaacagag ggcttaacaa cctcccgaaa 660 tcaacatatc aagattggat aacctacaat agattgagga gggatctcac actgactgta 720 ctcgacattg ctgctttctt tcccaactac gataaccgta gatatcccat tcaacccgtc 780 ggacaactca ctcgtgaagt gtacacagat ccccttatca acttcaaccc acagttacaa 840 agtgttgcac agctgccaac cttcaatgtg atggaaaact ctgccattag gaatccacat 900 ctgtttgaca tacttaacaa cttgactatc tttacggatt ggtttagtgt ggggaggaac 960 ttctattggg gaggtcacag agtgattagc tctcttatcg gaggtgggaa cattacttca 1020 ccaatctatg gacgtgaagc aaatcaagaa ccaccacgtt cattcacctt caatggaccc 1080 gttttcagaa cactgtccaa tccgactctg agactgttgc agcaaccttg gccagcacct 1140 cctttcaact tgaggggtgt agagggagtc gagttttcca cacctactaa cagtttcacc 1200 tagggggtc gtggaactgt tgattctctt actgaacttc ctccagagga caattctgtt 1260 cctccgaggg aaggctattc acaccgtttg tgtcatgcaa cttttgttca gaggtctggc 1320 acacctttct taacaactgg cgtcgttttc agctggacac ataggagtgc aaccctcacg 1380 aatactatcg atccagagag aatcaatcag attcctcttg ttaagggctt tagggtgtgg 1440 ggtgggacct cagtgataac tggtcccggt ttcaccggag gagacatact tagaaggaac 1500 acctttgggg atttcgtttc tcttcaagta aacatcaatt ctcctatcac ccagagatac 1560 agacttaggt ttagatatgc tagtagccgt gacgccagag tcatagtgtt gactggtgct 1620 gcatcaactg gtgttggtgg gcaagtttca gtgaacatgc cacttcaaaa gacaatggag 1680 ataggtgaga acttgacgtc cagaaccttc agatacactg actttctaa tcctttctct 1740 tttcgtgcta atccagacat cataggaatc agtgaacaac cgttgtttgg agctggttcc 1800 atttctagtg gagaattgta cattgacaag attgagatca tacttgccga tgccacgctc 1860 gaggctgaat ctgatctcga aaggtga 1887 <210> 34 <211> 628 <212> PRT <213> Bacillus thuringiensis <400> 34 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 35 <211> 3495 <212> DNA <213> artificial sequence <220> <223> Synthesized coding district <400> 35 atggacaaca atcccaatat caatgagtgc atcccataca actgcctttc aaatcccgag 60 gaagctcttac tggatggga aggattagc actggcaatt ccagcattga catttccttg 120 agtctcgttc aattccttgt gagcaacttt gtgcctggcg gagggttctt ggttggtctc 180 atagactttg tttgggggaat tgtaggacca tcacagtggg atgccttctt agtccagatt 240 gaacagctta tcaatgagag aatagctgag ttcgcaagaa atgctgcaat cgctaacttg 300 gagggactcg gcaacaactt caacatctac gtggaggctt tcaaagaatg ggaagaagat 360 cctaagaatc cagctactag gactagagtc atcgataggt ttaggattct tgatgggctg 420 ttggagcgtg acattccttc ctttaggatt agtggcttcg aggttcctct cctttctgtc 480 tatgctcaag cagccaatct ccatctcgcc attctccgtg attcagtaat cttcggagaa 540 agatggggtt tgacaacgat caatgtgaat gagaactaca acagattgat cagacacatt 600 gatgagtatg ctgatcattg tgctaacaca tacaacagag ggcttaacaa cctcccgaaa 660 tcaacatatc aagattggat aacctacaat agattgagga gggatctcac actgactgta 720 ctcgacattg ctgctttctt tcccaactac gataaccgta gatatcccat tcaacccgtc 780 ggacaactca ctcgtgaagt gtacacagat ccccttatca acttcaaccc acagttacaa 840 agtgttgcac agctgccaac cttcaatgtg atggaaaact ctgccattag gaatccacat 900 ctgtttgaca tacttaacaa cttgactatc tttacggatt ggtttagtgt ggggaggaac 960 ttctattggg gaggtcacag agtgattagc tctcttatcg gaggtgggaa cattacttca 1020 ccaatctatg gacgtgaagc aaatcaagaa ccaccacgtt cattcacctt caatggaccc 1080 gttttcagaa cactgtccaa tccgactctg agactgttgc agcaaccttg gccagcacct 1140 cctttcaact tgaggggtgt agagggagtc gagttttcca cacctactaa cagtttcacc 1200 tagggggtc gtggaactgt tgattctctt actgaacttc ctccagagga caattctgtt 1260 cctccgaggg aaggctattc acaccgtttg tgtcatgcaa cttttgttca gaggtctggc 1320 acacctttct taacaactgg cgtcgttttc agctggacac ataggagtgc aaccctcacg 1380 aatactatcg atccagagag aatcaatcag attcctcttg ttaagggctt tagggtgtgg 1440 ggtgggacct cagtgataac tggtcccggt ttcaccggag gagacatact tagaaggaac 1500 acctttgggg atttcgtttc tcttcaagta aacatcaatt ctcctatcac ccagagatac 1560 agacttaggt ttagatatgc tagtagccgt gacgccagag tcatagtgtt gactggtgct 1620 gcatcaactg gtgttggtgg gcaagtttca gtgaacatgc cacttcaaaa gacaatggag 1680 ataggtgaga acttgacgtc cagaaccttc agatacactg actttctaa tcctttctct 1740 tttcgtgcta atccagacat cataggaatc agtgaacaac cgttgtttgg agctggttcc 1800 atttctagtg gagaattgta cattgacaag attgagatca tacttgccga tgccacgctc 1860 gaggctgaat ctgatctcga aagggcacag aaagctgtaa acgcattgtt tacaagttct 1920 aatcaaatcg gactcaaaac cgatgttacg gactatcaca tagatagggt ttctaatctt 1980 gtggaatgtc tttcagatga gttttgttta gatgagaaga aagaactttc agaaaaggtc 2040 aagcacgcca aaagactgtc cgatgaaagg aatcccttc aagacccaaa cttcgtgga 2100 atcaataggc agctcgacag aggttggaga gggagcacag atatcaccat tcaaggagga 2160 gatgacgttt tcaaagagaa ctatgtcacc ttgttaggca cctttgatga gtgctatcca 2220 acttatctgt atcagaagat tgatgaatcc aagctgaagg cttacacaag atatcagctc 2280 agaggataca tcgaggactc ccaagatttg gagatatact tgattcgtta caatgcaaaa 2340 catgagaccg tgaatgttcc tggtactgga agtctctggc cactgtctgc tccgtcacct 2400 attgggaaat gtgcccatca ctcccaccat ttctcattgg acatagacgt tggctgcaca 2460 gatttgaatg aagatttggg tgtttgggtc atcttcaaga tcaaaactca agacggacac 2520 gctcgtttag gaacttaga gttcttgaa gagaagccct tggttgggga ggcacttgcc 2580 agagtaaga gagctgaaaa gaagtgaga gatagagggg aggaacttga gtgggagact 2640 aacattgtgt aaggaagc haaagaagc gtggatgctc tttcgtgaa ctctcagtat 2700 gataggttac aagxagacac spacatagca atgatacatg cagctgaca aagagtccat 2760 tctattcgtg agccattactt gccagaactt agtgtgatc ccggtgtcaa cgctgccatt 2820 ttcgaggaat tggaggag aatctttacg gcttcagcc tctatgacgc taggaatgtt 2880 atcaagaatg gtgatttca caatggccctc tcatgttgga atgtgaaagg tcatgttgat 2940 gtagaggagc aaaaacaatca ccgtagcgtg ctggttgtcc cagaatggga agccgaagta 3000 agccaagaag ttagagttg ccctggaaga ggctacattc tgcgtgtcac cgcttacaaa 3060 gaaggatatg gcgaagggtg cgtgactatt catgagattg agaacaatac tgacgaactt 3120 aagttttcaa actgcgtcga ggaggaagtg tatcctaca acacagtgac ttgtaatgac 3180 tatacagcaa cgcaagagga atacgagggg acatacacca gtcgtaatcg tggttatgat 3240 ggtgcttatg aaagcaattc atccgttcca gctgactatg ccagtgccta cgaagagaag 3300 gcttacacgg atggcagaag agataaccca tgtgagtcca acagaggtta tggtgattac 3360 actcctcttc cagctggtta cgtgactaaa gagttagagt actttccgga gactgataag 3420 gtttggattg aaatcggaga gacagaaggg acattcatag tagattcagt tgagcttctt 3480 ctcatggaag aatga 3495 <210> 36 <211> 1164 <212> PRT <213> Artificial sequence <220> <223> Translated from a synthetic coding region <400> 36 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 thirty Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser 625 630 635 640 Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Arg 645 650 655 Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu 660 665 670 Lys Lys Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp 675 680 685 Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln 690 695 700 Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly 705 710 715 720 Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Leu Gly Thr Phe Asp 725 730 735 Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu 740 745 750 Lys Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln 755 760 765 Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val 770 775 780 Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Pro Ser Pro 785 790 795 800 Ile Gly Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp 805 810 815 Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe 820 825 830 Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe 835 840 845 Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg 850 855 860 Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr 865 870 875 880 Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val 885 890 895 Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Ala Met Ile 900 905 910 His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro 915 920 925 Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu 930 935 940 Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val 945 950 955 960 Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys 965 970 975 Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val 980 985 990 Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro 995 1000 1005 Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr 1010 1015 1020 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp 1025 1030 1035 Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn 1040 1045 1050 Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Glu Glu Tyr 1055 1060 1065 Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr 1070 1075 1080 Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Glu 1085 1090 1095 Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser 1100 1105 1110 Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val 1115 1120 1125 Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile 1130 1135 1140 Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu 1145 1150 1155 Leu Leu Leu Met Glu Glu 1160 <210> 37 <211> 3495 <212> DNA <213> Artificial sequence <220> <223> Synthetic coding region <400> 37 atggataaca atccgaacat caatgaatgc atcccgtaca actgcctgag caacccggaa 60 gaagtgctgt tggatggaga acggatatca actggtaatt catcaattga tatttctctg 120 tcacttgttc agtttctggt atctaacttc gtcccaggcg gaggattcct ggttggatta 180 atagattttg tatggggaat agttggccct tctcaatggg atgcatttct agtacaaatt 240 gaacaattaa ttaatgaaag aatagctgaa tttgctagga atgctgctat tgctaattta 300 gaaggattag gaaacaattt caatatatat gtggaagcat ttaaagaatg ggaagaagat 360 cctaagaatc cagcaaccag gaccagagta attgatcgct ttcgtatact tgatgggcta 420 cttgaaaggg acattccttc gtttcgaatt tctggatttg aagtacccct tttatccgtt 480 tatgctcaag cggccaatct gcatctagct atattaagag attctgtaat ttttggagaa 540 agatggggat tgacaacgat aaatgtcaat gaaaactata atagactaat taggcatatt 600 gatgaatatg ctgatcactg tgcaaatacg tataatcggg gattaaataa tttaccgaaa 660 tctacgtatc aagattggat aacatataat cgattgcgga gagacttaac attgactgta 720 ttagatatcg ccgctttctt tccaaactat gacaatagga gatatccaat tcagccagtt 780 ggtcaactaa caagggaagt ttatacggac ccattaatta attttaatcc acagttacag 840 tctgtagctc aattacctac ttttaacgtt atggagaaca gcgcaattag aaatcctcat 900 ttattgata tattgaataa tcttacaatc tttacggatt ggtttagtgt tggacgcaat 960 ttttattggg gaggacatcg agtaatatct agccttatag gaggtggtaa cataacatct 1020 cctatatatg gaagagaggc gaaccaggag cctccaagat cctttacttt taatggaccg 1080 gtatttagga ctttatcaaa tcctacttta cgattattac agcaaccttg gccagcgcca 1140 ccatttaatt tacgtggtgt tgaaggagta gaattttcta cacctacaaa tagctttacg 1200 tatcgaggaa gaggtacggt tgattcttta actgaattgc cgcctgagga taatagtgtg 1260 ccacctcgcg aaggatatag tcatcgttta tgtcatgcaa cttttgttca aagatctgga 1320 acacctttt taacaactgg tgtagtattt tcttggacgc atcgtagtgc aactcttaca 1380 aatacaattg atccagagag aattaatcaa atacctttag tgaaaggatt tagagtttgg 1440 gggggcacct ctgtcattac aggaccagga tttacaggag gggatatcct tcgaagaaat 1500 acctttggtg attttgtatc tctacaagtc aatattaatt caccaattac ccaaagatac 1560 cgtttaagat ttcgttacgc ttccagtagg gatgcacgag ttatagtatt aacaggagcg 1620 gcatccacag gagtgggagg ccaagttagt gtaaatatgc ctcttcagaa aactatggaa 1680 ataggggaga acttaacatc tagaacattt agatataccg attttagtaa tcctttttca 1740 tttagagcta atccagatat aattgggata agtgaacaac ctctatttgg tgcaggttct 1800 attagtagcg gtgaacttta tatagataaa attgaaatta ttctagcaga tgcaacattt 1860 gaagcagaat ctgatttaga aagagcacaa aaggcggtga atgccctgtt tacttcttcc 1920 aatcaaatcg ggttaaaaac cgatgtgacg gactatcata tcgatcgagt atccaattta 1980 gttgagtgtt tatctgatga atttgtctg gatgaaaaaa aagaattgtc cgagaaagtc 2040 aaacatgcga agcgacttag tgatgagcgg aatttacttc aagatccaaa ctttagaggg atcaatagac aactagaccg tggctggaga ggaagtacgg atattaccat ccaaggaggc gatgacgtat ccaaagagaa ttacgttacg ctattgggta cctttgatga gtgctactta acgtatttat atcaaaaaat agatgagtcg aaattaaaag cctatacccg ttaccaatta aggggtata tcgaagatag tcaagactta gaatctatt taattcgcta caatgccaaa 2400. ccgaaacag taaatgtgcc aggtacgggt tccttatggc cgctttcagc cccaagtcca atcggaaaat gtgcccatca ttcccatcat ttctccttgg acattgatgt tggatgtaca gacttaaatg aggacttagg tgtatgggtg atattcaaga ttaagacgca agatggccat gcaagactag gaaatctaga atttctcgaa gagaaaccat tagtagga agcactagct cgtgtgaaaa gagcggaga aaaatggaga gacaaacgtg aaaaattgga atgggaaaca aatattgttt ataaagaggc aaaagaatct gtagatgctt tatttgtaaa ctctcaatat gatagattac aagcggatac caacatcgcg atgattcatg cggcagataa acgcgttcat agcattcgag aagcttatct gcctgagctg tctgtgattc cgggtgtcaa tgcggctatt 2820 tttgaagaat tagaagggcg tattttcact gcattctccc tatatgatgc gagaaatgtc 2880 attaaaatg gtgattttaa taatggctta tcctgctgga acgtgaaagg gcatgtagat 2940 gtagaaagc aaaacaacca ccgttcggtc cttgttgttc cggaatggga agcagaagtg 3000 tcacaagaag ttcgtgtctg tccgggtcgt ggctatatcc ttcgtgtcac agcgtacaag 3060 gagggatatg gagaaggttg cgtaaccatt catgagatcg agaacaatac agacgaactg 3120 aagtttagca actgtgtaga agagaagta tatccaaaca acacggtaac gtgtaatgat 3180 tatactgcga ctcaagaaga atatgagggt acgtacactt ctcgtaatcg aggatatgac 3240 ggagcctatg aaagcaattc ttctgtacca gctgattatg catcagccta tgaagaaaaa 3300 gcatatacag atggacgaag agacaatcct tgtgaatcta acagaggata tggggattac 3360 acaccactac cagctggcta tgtgacaaaa gaattagagt acttcccaga aaccgataag 3420 gtatggattg agatcggaga aacggagagga acattcatcg tggacagcgt ggaattactt 3480 cttatggagg aataa 3495 <210> 38 <211> 1164 <212> PRT <213> Artificial sequence <220> <223> Translation from the synthetic coding region <400> 38 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser 625 630 635 640 Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Arg 645 650 655 Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu 660 665 670 Lys Lys Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp 675 680 685 Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln 690 695 700 Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly 705 710 715 720 Asp Asp Val Ser Lys Glu Asn Tyr Val Thr Leu Leu Gly Thr Phe Asp 725 730 735 Glu Cys Tyr Leu Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu 740 745 750 Lys Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln 755 760 765 Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val 770 775 780 Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Pro Ser Pro 785 790 795 800 Ile Gly Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp 805 810 815 Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe 820 825 830 Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe 835 840 845 Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg 850 855 860 Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr 865 870 875 880 Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val 885 890 895 Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Ala Met Ile 900 905 910 His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro 915 920 925 Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu 930 935 940 Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val 945 950 955 960 Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys 965 970 975 Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val 980 985 990 Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro 995 1000 1005 Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr 1010 1015 1020 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp 1025 1030 1035 Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn 1040 1045 1050 Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Glu Glu Tyr 1055 1060 1065 Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr 1070 1075 1080 Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Glu 1085 1090 1095 Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser 1100 1105 1110 Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val 1115 1120 1125 Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile 1130 1135 1140 Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu 1145 1150 1155 Leu Leu Leu Met Glu Glu 1160 <210> 39 <211> 2109 <212> DNA <213> Artificial sequence <220> <223> Synthetic coding region <400> 39 atggcacaga gcagtaggat ctgccacggt gtgcagaacc cgtgcgtgat catctcgaac 60 ctgagcaagt ccaaccagaa caagtcaccg ttctccgtgt ccctcaagac ccaccagcac 120 ccgagagcct acccgatcag cagctcctgg ggactgaaga agagtggcat gaccctgatc ggctccgagc tgagaccgct gaaggtgatg tccagcgtgt caagcggataa caacccgaac atcaacgagt gcatccccta caactgcctg agcaacccg aggaggtgct gctggacggc 300 gagaggatct caaccggcaa cagcagcatc gacatcagcc tgtccctggt gcagttcctg gtgagcaact tcgtgccgggg cggcggcttc ctggtgggat taatcgactt cgtgtggggc 420 atcgtcggcc cgtcccagtg ggacgccttc ctggttcaga tcgagcaatt aattaatgaa 480 aggatagcg agttcgcgag gaacgcggcc atcgccaacc tggagggcct gggcaacaac 540. ttcaacatct acgtggaagc atttaaggag tgggaggagg accccaagaa cccggccacg aggacgaggg tgatcgaccg ctttcgcatc ctggacggcc tgctggagag ggacatcccg 660 tccttcagaa tcagcggctt cgaggtcccg ctgctgtccg tgtacgcgca agcggccaac 720 ctgcacctgg cgatcctgag ggactccgtg atattcggcg agaggtgggg cctgaccacc 780 atcaacgtga atgaaaacta caaccggctg ataaggcaca tcgacgagta cgccgaccac tgcgccaaca cctacaatag gggattaaat aatctgccca agagcaccta ccaagactgg 900 atcacatata accggctgag gagggacctg accctgaccg tgctggacat cgccgcgttc 960 ttcccgaact acgacaatag gcgctacccg atccagccgg tgggccagct gacccgcgag 1020 gtgtacaccg acccattaat taatttcaac ccgcagctcc agtccgtggc ccagctgccg 1080 accttcaacg tgatggagaa cagcgccatc cggaacccgc acctgttcga catcctgaat 1140 aatctgacca tcttcaccga ctggttctca gtgggccgga acttctactg gggcggccat 1200 agggtgatct ccagcctgat cggcggcggc aacatcacct ccccgatcta cgggagggag 1260 gcgaaccagg agccgccgag gtccttcacc ttcaacggcc cggtgtttag gaccctgtcc 1320 aacccgaccc tgaggctgct ccagcagccg tggccggcgc cgccgttcaa cctgaggggc 1380 gtggagggcg tggagttcag caccccgacc aacagcttca cctaccgggg gaggggcacc 1440 gtggactcac tgaccgagct gccgccggag gacaacagcg tgccgccgag ggagggctac 1500 agccataggc tgtgccacgc caccttcgtg cagaggagcg gcaccccgtt cttgacgacc 1560 ggcgtggtgt tctcctggac ccaccggagc gcgaccctga ccaatacaat cgacccggag 1620 ggcgtggtgt tctcctggac ccaccggagc gcgaccctga ccaatacaat cgacccggag 1620 agaattaatc aaatcccgct ggtgaagggc ttccgggtgt ggggcggcac ctccgtgatc 1680 agaattaatc aaatcccgct ggtgaagggc ttccgggtgt ggggcggcac ctccgtgatc 1680 accgggccgg gctttaccgg cggcgacatc ctgaggagga acacgttcgg cgacttcgtg 1740 accgggccgg gctttaccgg cggcgacatc ctgaggagga acacgttcgg cgacttcgtg 1740 agcctccaag tgaacattaa tagcccgatc acccagcgct accggctgag gttccgctac 1800 agcctccaag tgaacattaa tagcccgatc acccagcgct accggctgag gttccgctac 1800 gcgtcaagcc gcgacgcgag ggtgatcgtg ctgaccggcg cggcctcaac cggcgtgggc 1860 gcgtcaagcc gcgacgcgag ggtgatcgtg ctgaccggcg cggcctcaac cggcgtgggc 1860 ggccaagtgt ccgtgaacat gccgctgcaa aagacgatgg agatcggcga gaacctgacc 1920 ggccaagtgt ccgtgaacat gccgctgcaa aagacgatgg agatcggcga gaacctgacc 1920 tcaaggacct tccgctacac cgacttcagc aacccgttca gctttagggc caacccggat 1980 tcaaggacct tccgctacac cgacttcagc aacccgttca gctttagggc caacccggat 1980 ataatcggca tcagcgagca gccgctgttc ggcgccggct ccatctcaag cggcgagctg 2040 ataatcggca tcagcgagca gccgctgttc ggcgccggct ccatctcaag cggcgagctg 2040 tacatcgata aaatcgagat catcctggcg gacgcgacct tggaggccga gtccgacctg 2100 tacatcgata aaatcgagat catcctggcg gacgcgacct tggaggccga gtccgacctg 2100 gagaggtga 2109 gagaggtga 2109 <210> 40<210> 40 <211> 702 <211> 702 <212> PRT <212> PRT <213> 人工序列 <213> Artificial sequence <220> <220> <223> 自合成的编码区翻译的 <223> Translated from a synthetic coding region <400> 40 <400> 40 Met Ala Gln Ser Ser Arg Ile Cys His Gly Val Gln Asn Pro Cys Val Met Ala Gln Ser Ser Arg Ile Cys His Gly Val Gln Asn Pro Cys Val 1 5 10 15 Ile Ile Ser Asn Leu Ser Lys Ser Asn Gln Asn Lys Ser Pro Phe Ser 20 25 30 Val Ser Leu Lys Thr His Gln His Pro Arg Ala Tyr Pro Ile Ser Ser 35 40 45 Ser Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu 50 55 60 Arg Pro Leu Lys Val Met Ser Ser Val Ser Ala Asp Asn Asn Pro Asn 65 70 75 80 Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val 85 90 95 Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile 100 105 110 Serum Leu Serum Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly 115 120 125 Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro 130 135 140 Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Glu 145 150 155 160 Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly 165 170 175 Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu 180 185 190 Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr Arg Val Ile Asp Arg Phe 195 200 205 Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile 210 215 220 Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Ala Asn 225 230 235 240 Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp 245 250 255 Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg 260 265 270 His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly 275 280 285 Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp Ile Thr Tyr Asn 290 295 300 Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe 305 310 315 320 Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro Val Gly Gln 325 330 335 Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gln 340 345 350 Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met Glu Asn Ser 355 360 365 Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile 370 375 380 Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His 385 390 395 400 Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile 405 410 415 Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe Thr Phe Asn 420 425 430 Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gln 435 440 445 Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val 450 455 460 Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr 465 470 475 480 Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro 485 490 495 Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gln Arg 500 505 510 Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His 515 520 525 Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gln 530 535 540 Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile 545 550 555 560 Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe 565 570 575 Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro Ile Thr Gln 580 585 590 Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val 595 600 605 Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gln Val Ser 610 615 620 Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn Leu Thr 625 630 635 640 Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg 645 650 655 Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala 660 665 670 Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile 675 680 685 Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser Asp Leu Glu Arg 690 695 700 BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Shown are the levels of expression of DIG-465 by construct 115752 and DIG-473 by construct 115753 in T1 maize leaves sampled by leaf punch.

[0068] Figure 2 is a plot of the amount of leaf damage caused by FAW or Cry1Fa-resistant FAW in maize versus the expression level of DIG-465.

[0069] Figure 3 The amount of leaf damage caused by FAW or Cry1Fa-resistant FAW in maize is plotted against the expression level of DIG-473.

[0070] Detailed Description of the Invention

[0071] The term "genetic material" as used herein is intended to include all genes, nucleic acids, DNA, and RNA. These sequences have been altered in a manner to improve the stability of the expressed protein toxin when the gene is transformed into plants, particularly corn and dicots. The protein toxins discussed herein are generally referred to as "insecticides" or "insecticides." Insecticides and "insecticides" herein mean that the protein toxins have "functional activity" as further defined herein and are used as insect control agents.

[0072] "Functionally active" as used herein means that the protein toxins can function as insect control agents because the proteins are orally active, or have toxic effects, or are capable of disrupting or preventing feeding, which may or may not result in insect mortality. When insects come into contact with an effective amount of the toxins delivered via transgenic plant expression, formulated protein compositions, sprayable protein compositions, bait matrices, or other delivery systems, the result is typically insect mortality or the insects' inability to feed on the source that provided the toxin to the insects.

[0073] The term "oligonucleotide" refers to a macromolecule composed of a short chain of nucleotides of RNA or DNA. Such length can be at least one nucleotide, but is generally in the range of about 10 to about 12 nucleotides. Determination of the length of an oligonucleotide is within the skill of the artisan and should not be construed as limiting herein. Thus, an oligonucleotide can be less than 10 or greater than 12. The subject invention relates not only to polynucleotide sequences encoding these types of toxins, but also to the use of these polynucleotide sequences for producing recombinant hosts expressing the toxins.

[0074] As used herein, the term "toxic" means that the toxin produced by Bacillus thuringiensis has "functional activity" as defined herein.

[0075] The term "modified CrylCa toxin" is intended to include all protein sequences of the Sequence Listing and all variants thereof described herein.

[0076] As used herein, the term "genetic material" is intended to include all genes, nucleic acids, DNA and RNA.

[0077] For the nomenclature of nucleotide residues of polynucleotides, DNA, RNA, oligonucleotides and primers, and for the nomenclature of amino acid residues of proteins, standard IUPAC abbreviations are used herein. Nucleic acid sequences are presented in the standard 5' to 3' orientation, and protein sequences are presented in the standard amino (N) terminus to carboxyl (C) terminus orientation.

[0078] The toxins and genes of the present invention can be further defined by their amino acid and nucleotide sequences, as well as the sequences of unique fragments contained within the full-length DNA amino sequence. The sequences of molecules within each new class can be defined by their homology to specific exemplary sequences, as well as their ability to hybridize with or be amplified by certain exemplary probes and primers. The toxin classes provided herein can also be identified based on their immunoreactivity with certain antibodies.

[0079] Toxin structure.The toxins of the subject invention can also be characterized based on their structure and domain composition. Due to the correlation between protein sequence variability and differences in the spectrum of biological activities, it has been hypothesized that the "hypervariable region" between blocks three and five underlies the differences in insect specificity among various Bt delta-endotoxins.

[0080] When the gene encoding the native full-length Cry1Ca protein was inserted into corn cells and expressed in corn cells, at least five detectable proteolytic degradation products were observed. These five polypeptides were determined to have the following amino acid lengths: 1-1164, 1-628, 29-628, 74-628, and 74-596. Of the five Cry1Ca degradation products detected, two fragments were found to be inactive against key driver insect pests. In most cases, these two inactive fragments represented the major portion of the Cry1Ca-related protein detected in corn cells. Expressing the native full-length Cry1Ca gene in corn resulted in plants with insufficient functional activity against key pests such as the fall armyworm.

[0081] When a gene expressing a truncated native Cry1Ca protein (aa 1-628) is inserted into and expressed in corn cells, less proteolytic processing occurs. Most of it remains unprocessed and functionally active. Therefore, after expressing the truncated Cry1Ca gene in corn cells, the plant has sufficient functional activity against major insect pests due to reduced proteolysis in the corn cells.

[0082] Alterations to the original amino acid sequence of Cry1Ca offer the potential for achieving continuous bioactivity against key insect pests and result in reduced proteolytic processing of the protein, as measured in vitro using chymotrypsin as a protease. Reduced proteolytic processing of the altered Cry1Ca protein results in higher amounts of functionally active protein accumulating in plants and greater activity against target insect pests.

[0083] Protease-sensitive variants.Insect gut proteases generally help insects obtain the amino acids they need from their prey proteins. The best-understood insect digestive proteases are serine proteases, which appear to be the most common type (Englemann and Geraerts, 1980), particularly in Lepidoptera species. The gut of Coleopteran insects is more neutral to acidic than that of Lepidoptera. Most Coleopteran larvae and adults, such as the Colorado potato beetle, have a slightly acidic midgut, with cysteine ​​proteases providing the primary proteolytic activity (Wolfson and Murdock, 1990). More specifically, Thie and Houseman (1990) identified and characterized cysteine ​​proteases—cathepsin B-like and cathepsin H-like proteases—and an aspartyl protease—cathepsin D-like protease in the Colorado potato beetle. Gillikin et al. (1992) characterized the proteolytic activity in the gut of western corn rootworm larvae and found that cysteine ​​proteases were predominantly involved. U.S. Patent No. 7,230,167 discloses the presence of the serine protease cathepsin G in western corn rootworm. The diversity and different activity levels of insect gut proteases may affect insect sensitivity to specific Bt toxins.

[0084] In one embodiment, toxin has specific change in its amino acid sequence, thereby significantly reduces the protease processing level of the protein of the naturally occurring protease in corn plant to the protein of expression.These amino acid whose variation causes this protein functional activity level when expressing in corn is higher.Can introduce protease cleavage site (Horton etc., 1989) at the desired position by chemical gene synthesis or splicing overlapping PCR.For example, can randomly insert serine protease recognition sequence to influence the protein processing at the deletion site of the expectation in some insect pest midgut at the specific site in Cry protein structure.By engineering protease recognition sequence at the processing site of expectation, can utilize Lepidoptera midgut serine protease, as trypsin or trypsin sample enzyme, chymotrypsin, elastase etc. (Christler etc., 1992) activate Cry albumen.Equally, can similarly utilize Coleopteran serine protease, as trypsin, chymotrypsin and cathepsin G sample protease by engineering recognition sequence at the processing site of expectation. In addition, by engineering recognition sequences at the desired processing sites, coleopteran cysteine ​​proteases such as cathepsins (B-like, L-like, O-like, and K-like proteases) (Koiwa et al., 2000 and Bown et al., 2004), metalloproteases such as ADAM10 (Ochoa-Campuzano et al., 2007), aspartic proteases such as cathepsin D-like and E-like proteases, pepsin, plasmepsin, and chymosin can be utilized.

[0085] The scope of the present invention includes variant Cry1Ca insecticidal proteins, which are produced by manipulating the coding sequence of the insecticidal protein of the present invention, introducing or eliminating protease processing sites at appropriate positions to allow or eliminate insect, plant or microbial proteases to proteolytically cleave larger variant proteins. The final result of this operation is to produce toxin molecules with the same or better activity as the complete (full-length) natural toxin protein.

[0086] Different from the DNA substrate recognition and the high sequence specificity in cutting of II type restriction endonucleases, proteolytic enzymes are more non-specific to the amino sequence comprising the cutting recognition site. Compared with cathepsin B, K, L and S (Bown et al., 2004), it has been found that some commonalities of the amino acid structure comprising some protease cleavage sites, particularly cathepsin G, have been found. In the nomenclature of the protease cleavage site in the following illustration, the amino acid residues upstream (i.e., towards the N-terminal) of the cleavage site are numbered P1, P2, P3, P4, P5 etc., and residue P1 is next to the cleavage site, and residue P5 is the fifth farthest from the cleavage site in the N-terminal direction. The amino acid residues downstream (i.e., towards the C-terminal) of the cleavage site are numbered P1 ', P2 ', P3 ', P4 ', P5 ' etc., and residue P1 ' is next to the cleavage site, and residue P5 ' is the fifth farthest from the cleavage site in the C-terminal direction. Known cathepsin G shows preferential cleavage after the P1 residue glutamine, lysine, tryptophan or phenylalanine, wherein residues P2, P3, P4, P5 etc., as well as P1 ', P2 ', P3 ', P4 ', P5 ' etc., can be any of the 20 kinds of amino acids commonly found in natural proteins. Cathepsin B, K, L and S show a little enhanced cleavage site sequence specificity, wherein the side chain of the P2 amino acid fits into the substrate binding site S2 of cathepsin. The S2 site of these cathepsins preferentially interacts with the P2 amino acid with a large hydrophobic side chain (e.g., found in valine, leucine, isoleucine, phenylalanine, tryptophan and tyrosine), and is unfavorable for interacting with the P2 residue with a charged side chain (except that cathepsin B and L accept the large hydrophilic charged side chain of the arginine on the P2 position). Some specificity can be seen in the identity of the amino acid at the P3 position. For example, when phenylalanine or arginine occupies the P2 position, cathepsin L preferentially cuts after the arginine at the P1 position. The P3 amino acid can be aromatic (e.g., phenylalanine, tryptophan, histidine, or tyrosine) or hydrophobic (e.g., alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, or tyrosine). Positions P4, P5, etc., and P1', P2', P3', P4', P5', etc., can be any of the 20 amino acids commonly found in natural proteins.

[0087] Proteolytic cleavage also depends on the availability of the subject cleavage sequence to various proteases; potential cleavage sites sequestered within the three-dimensional structure of the protein may render the protein resistant to cleavage by a particular protease. It has been suggested that the diversity and varying activity levels of insect gut proteases may influence insect sensitivity to specific Bt toxins. Those skilled in the art of biochemistry and molecular biology can assay for the biochemical characteristics (including, but not limited to, determining the amino acid sequences comprising the N- and C-termini of the polypeptide) of insecticidal protein fragments produced by protease cleavage / activation of larger proteins by intestinal proteases of susceptible insects. It is also possible to characterize the protease systems of the gut or host plant of non-susceptible insects and construct sequences within the coding sequence of the Bt insecticidal protein that are readily cleaved by proteases in the gut of non-susceptible insects or in the host plant in which the Bt insecticidal protein is intended to be produced transgenically. Such analyses and manipulations of the subject Bt insecticidal proteins are understood to fall within the scope of the present invention.

[0088] In another embodiment, the toxin has specific changes in its amino acid sequence that significantly increase the protein expression level of the toxin when expressed in a variety of different expression systems (including plants and bacteria). The result of increased protein expression is increased functional activity in the expression system. The advantage is that high doses of toxin are provided to insects, which can prevent the insects from developing resistance to the toxin due to the survival of small insect populations that receive sublethal doses of the protein toxin.

[0089] Genes and toxins The protein molecules of the embodiments herein comprise amino acid sequences homologous to known insecticidal proteins, particularly BtCry proteins, more particularly CrylCa protein (Genbank Accession No.: AAA22343). The predicted amino acid sequences encoded by the nucleotide sequences of these embodiments are disclosed as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

[0090] The toxin sequences of the subject invention are provided as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. In preferred embodiments, the toxins of the invention have at least one of the following characteristics:

[0091] (a) The toxin is encoded by a nucleotide sequence that hybridizes under stringent conditions to a sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or a complementary sequence thereof.

[0092] (b) the toxin is immunoreactive with antibodies raised against an insecticidal toxin of about 68-71 kDa from an isolate of Bacillus thuringiensis or a fragment thereof.

[0093] (c) the toxin is encoded by a nucleotide sequence, a portion of which can be amplified by PCR using a primer pair to produce a fragment of about 25-40 bp,

[0094] (d) the toxin comprises an insecticidal portion of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

[0095] (e) the toxin comprises an amino acid sequence having at least about (90%) homology to an insecticidal portion of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40,

[0096] (f) the toxin is encoded by a nucleotide sequence that hybridizes under stringent conditions to an insecticidal portion of a nucleotide sequence selected from the group consisting of a DNA encoding SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40,

[0097] (g) the toxin is immunoreactive with antibodies directed against the approximately 68 kDa or 130 kDa insecticidal toxin from Bacillus thuringiensis isolate MR-1206, or a fragment thereof.

[0098] (h) the toxin comprises an amino acid sequence that is at least about (90%) homologous to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40, and an insecticidal portion of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

[0099] The specific genes exemplified herein, variants of these genes, and fragments of these genes can also be obtained by synthetic construction, for example, by the method currently implemented by any one of several commercial suppliers (see, for example, U.S. Patent No. 7482119). These genes or parts or variants thereof can also be synthetically constructed, for example, by using a gene synthesizer and, for example, the method of U.S. Patent No. 5,380,831. Alternatively, standard molecular biology techniques for making point mutations can be used to easily construct variants of synthetic or naturally occurring genes. Fragments of these genes can also be prepared using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or by site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. In addition, gene fragments encoding active toxin fragments can be obtained using a variety of restriction enzymes.

[0100] , 400, 500, 600, 750, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 3000, 3500 nucleotides, or up to the number of nucleotides present in the nucleotide sequence encoding the insecticidal toxin disclosed and claimed herein (e.g., 1,878 nucleotides for SEQ ID NO: 1; 1,878 nucleotides for SEQ ID NO: 2). Fragments of a nucleotide sequence encoding a biologically active portion of a claimed protein will encode at least about 15, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, or 1200 contiguous amino acids, or up to the total number of amino acids present in the full-length pesticidal protein of the invention (e.g., 625 amino acids for SEQ ID NO: 2, or 1164 amino acids for SEQ ID NO: 38).

[0101] Recombinant host.The toxin encoding genes of the present invention can be introduced into a wide variety of microorganisms or plant hosts. The expression of the toxin gene directly or indirectly leads to the intracellular production and maintenance of the insecticidal protein. With a suitable microbial host, such as Pseudomonas, the microorganisms can be applied to the environment of the pest, where they will reproduce and may be ingested. The result is control of the pest. Alternatively, the microorganism containing the toxin gene can be treated under conditions that prolong the toxin activity and stabilize the cells. The treated cells that retain the toxin activity can then be applied to the environment of the target pest.

[0102] In cases where toxin genes are introduced into microbial hosts via suitable vectors and the hosts are applied to the environment in a living state, it is necessary to use certain host microorganisms. Microbial hosts are selected that are known to occupy the "phyllosphere" (rachis, phyllosphere, rhizosphere, and / or rhizosphere) of one or more crops of interest. These microorganisms are selected so that they can successfully compete with wild-type native microorganisms in specific environments (crops and other insect habitats), provide stable maintenance and expression of the gene expressing the polypeptide insecticide, and desirably provide improved protection of the insecticide from environmental degradation and inactivation.

[0103] Bt spores or recombinant host cells can also be treated before application or formulation for application to plants. For example, isolated Bt spore toxin crystals can be chemically treated to prolong insecticidal activity to include the treated polypeptide of the present invention (U.S. Patent No. 4,695,462 and Gaertner et al., 1993).

[0104] A variety of microorganisms are known to inhabit the rhizosphere (the surface of plant leaves) and / or the root zone (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms such as bacteria, for example, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Sinorhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, and the like. bacter), Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; and fungi, particularly yeasts, such as Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.Of particular interest are phyllospheric bacterial species such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Rhodopseudomonas spheroides, Xanthomonas campestris, Sinorhizobium meliloti (formerly Rhizobium meliloti), Alcaligenes eutrophus, and Azotobacter vinieriensis. vinelandii); and phyllosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, Saccharomyces cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are colored microorganisms.

[0105] A preferred embodiment of the present invention involves transforming plants with genes encoding the insecticidal proteins of the present invention or variants thereof. Transformed plants are resistant to attack by target insect pests due to the presence of controlled amounts of the subject insecticidal proteins or variants thereof in the cells of the transformed plants. By incorporating genetic material encoding the insecticidal properties of the Bt insecticidal toxin into the genome of plants that are preyed upon by specific insect pests, the adults or larvae die after consuming the food plants. Many members of the monocot and dicot taxa have been transformed. Transgenic crops, as well as fruits and vegetables, are of commercial interest. These crops include, but are not limited to, corn, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like. Several techniques exist for introducing foreign genetic material into monocot or dicot plant cells and for obtaining fertile plants that stably maintain and express the introduced genes. These techniques include direct accelerated delivery of genetic material encapsulated in microparticles into cells (U.S. Patents 4,945,050 and 5,141,131). Plants can be transformed using Agrobacterium technology, see U.S. Patent No. 5,177,010, U.S. Patent No. 5,104,310, European Patent Application No. 0,131,624, B1, European Patent Application No. 120,516, European Patent Application No. 159,418, B, European Patent Application No. 176,112, U.S. Patent No. 5,149,645, U.S. Patent No. 5,469,976, U.S. Patent No. 5,464,763, U.S. Patent No. 4,940,838, U.S. Patent No. 4,693,976, European Patent Application No. 116,718, European Patent Application No. 290,799, European Patent Application No. 320,500, European Patent Application No. 604,662, European Patent Application No. 627,752, European Patent Application No. 0,267,159, European Patent Application No. 0,292,435, U.S. Patent No. 5,231,019, U.S. Patent No. 5,463,174, U.S. Patent No. 4,762,785, U.S. Patent No. 5,004,863, and U.S. Patent No. 5,159,135. Other transformation techniques include WHISKERS TM Technology is shown in U.S. Patent No. 5,302,523 and U.S. Patent No. 5,464,765. Electroporation technology has also been used to transform plants, see WO 87 / 06614, U.S. Patent No. 5,472,869, U.S. Patent No. 5,384,253, WO9209696, and WO9321335. All of these patents and publications for transformation are incorporated herein by reference. In addition to many technologies for transforming plants, the type of tissue contacted with the foreign gene can also vary. Such tissue will include, but is not limited to, embryogenesis tissue, type I and type II callus, hypocotyl, meristem, etc. Almost all plant tissues can be transformed using appropriate techniques within the capabilities of those skilled in the art during dedifferentiation.

[0106] Genes encoding modified Cry1Ca insecticidal toxins and variants can be inserted into plant cells using various techniques well known in the art. For example, a variety of cloning vectors containing markers that allow selection of transformed microbial cells and functional replication systems in E. coli can be used to prepare and modify exogenous genes for insertion into higher plants. Such manipulations can include, for example, insertion of mutations, truncations, additions, deletions, or substitutions, depending on the desired application. Vectors include, for example, pBR322, the pUC series, the M13mp series, and pACYC184. Thus, the sequence encoding the Cry protein or variant can be inserted into appropriate restriction sites within the vector. The resulting plasmid is used to transform E. coli, the cells are cultured in a suitable nutrient medium, and then harvested and lysed to recover a usable amount of plasmid. Sequence analysis, restriction fragment analysis, electrophoresis, and other biochemical and molecular biological methods are commonly used as analytical methods. After each manipulation, the DNA sequence used can be cut and ligated to the next DNA sequence. Each manipulated DNA sequence can be cloned into the same or another plasmid.

[0107] Depending on the plant transformation method, auxiliary DNA sequences may be necessary. If, for example, a Ti or Ri plasmid is used to transform a plant cell, at least the T-DNA right border repeat sequence, but usually the right border repeat sequence and the left border repeat sequence of the Ti or Ri plasmid will be joined as the flanking regions of the gene to be inserted into the plant cell. The use of T-DNA-containing vectors for plant cell transformation has been extensively studied and fully described in EP 120516; Lee and Gelvin (2008), Fraley et al., (1986) and An et al., (1985) are well established in the field.

[0108] Once the inserted DNA has been integrated into the plant genome, it is relatively stable in all subsequent generations. The vectors used to transform plant cells usually contain a selectable marker gene that encodes a protein that confers tolerance to herbicides or antibiotics (such as bialaphos, kanamycin, G418, bleomycin or hygromycin) or the like on the transformed plant cells. Therefore, the selectable marker gene used alone should accordingly allow the selection of transformed cells, while the growth of cells that do not contain the inserted DNA is inhibited by the selection compound.

[0109] There are multiple techniques that can be used to insert DNA into host plant cells. These techniques include T-DNA transformation using Agrobacterium tumefaciens or Agrobacterium rhizogenes. In addition, fusion of plant protoplasts with liposomes containing the DNA to be delivered, direct injection of DNA, biolistic transformation (microparticle bombardment) or electroporation, and other possible methods can be used. Those skilled in the art of plant transformation will appreciate that there are multiple methodologies that can be used to produce transformed plants, and that they can be modified and specialized to adapt to the biological differences between various host plant species.

[0110] If using Agrobacterium strain to transform, the DNA to be inserted is cloned into a special plasmid, i.e., either into an intermediate (shuttle) vector or into a binary vector. The intermediate vector can be integrated into the Ti or Ri plasmid or its derivative by homologous recombination, using sequences homologous between the Ti or Ri plasmid and the intermediate plasmid. The Ti or Ri plasmid also comprises a vir region, which contains the vir genes required for transferring the T-DNA. The intermediate vector cannot be replicated in Agrobacterium. The intermediate vector can be transferred to Agrobacterium tumefaciens by chemically mediated transformation or other methods, using a helper plasmid (engaging through bacteria), electroporation, direct DNA, or other methods. Binary vectors can replicate autonomously in both Escherichia coli and Agrobacterium cells. They include sequences framed by right and left T-DNA border repeats, which can include sequences of selectable marker genes, cloning junctions, cloning polylinkers, or other sequences that can serve as introduction sites for genes for plant cell transformation in the plant cells being selected for transformation. They can be directly transformed into agrobacterium cells (Holsters et al., (1978)) by electroporation, or by direct DNA, chemically mediated conversion or by bacterial conjugation or other methodologies. The agrobacterium used as the host cell will comprise a plasmid carrying the vir region. The vir region is necessary for T-DNA to be transferred into the plant cell. In addition to the T-DNA region encoding the Bt insecticidal toxin protein or its variant, other T-DNA regions may be present in the agrobacterium host cell. The bacterial cells thus transformed are used for the transformation of plant cells. Advantageously, plant explants (for example, fragments of leaves, sections of stems, roots, and protoplasts or suspension cultured cells) can be cultivated together with agrobacterium tumefaciens or agrobacterium rhizogenes to transfer DNA into the plant cell. Then, after implanting suitable growth conditions and culture medium, whole plants can be regenerated from infected plant material, wherein the culture medium may contain antibiotics or herbicides for selecting transformed plant cells. Then, the plant thus obtained can be tested for the presence or absence of inserted DNA.

[0111] The transformed cells grow in the plant in the usual way. They can form reproductive cells and pass the transformed traits to subsequent generations of plants. Such plants can be cultivated in the normal way and hybridized with plants that have the same transformed genetic factors or other genetic factors. The resulting hybrids have corresponding phenotypic properties, such as the ability to control feeding of plant pests.

[0112] In those cases where injection and electroporation are used, there are no particular requirements for plasmid construction. Conventional plasmids, such as appropriately modified pUC derivatives containing all the genes desired to be transferred into plant cells, can be used.

[0113] The activity of the recombinant polynucleotide inserted into the plant cell can depend on the influence of the endogenous plant DNA adjacent to the insert. Therefore, another option is to utilize events that are known to be excellent insertion locations in the plant genome. See, for example, WO 2005 / 103266 A1, which relates to Cry1F and CrylAc cotton events; the subject Bt insecticidal toxin gene can replace Cry1F or CrylAc inserts in those genomic loci. According to the present invention, for example targeted homologous recombination can be used. This type of technology is the subject of, for example, WO03 / 080809 and corresponding disclosed U.S. applications (USPA 20030232410), which relate to using zinc fingers to carry out targeted recombination. It is also known in the art to use recombinases (such as cre-lox and flp-frt).

[0114] In a preferred embodiment of the present invention, plants will be transformed with genes in which the codon usage of the protein coding region has been optimized for plants. See, for example, U.S. Patent No. 5,380,831, which is incorporated herein by reference. In addition, advantageously, plants encoding truncated toxins will be used. Truncated toxins will typically encode about 55% to about 80% of the full-length toxin. Methods for creating synthetic Bt genes for plants are known in the art (Stewart 2007).

[0115] Another variable is the choice of selectable marker. The preference for a specific marker is at the discretion of those skilled in the art, but any of the following selectable markers, along with any other genes that may be used as selectable markers not listed herein, may be used. These selectable markers include, but are not limited to: the aminoglycoside phosphotransferase gene of the transposon Tn5 (Aph II), which encodes resistance to the antibiotics kanamycin, neomycin, and G418; and genes encoding tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos); imidazolinone, sulfonylurea, and triazolopyrimidine herbicides, such as chlorsulfuron, bromoxynil, and darabond. Examples of these genes are provided in Merlo (2002), which is incorporated herein by reference.

[0116] In addition to the selective marker, a reporter gene can be used. In some cases, a reporter gene can be used without a selective marker. A reporter gene is a gene that does not generally provide a growth advantage to the recipient organism or tissue. Reporter genes generally encode proteins that can provide certain phenotypic changes or enzymatic properties. A preferred reporter gene is the glucuronidase (GUS) gene. Other examples of reporter genes are provided in Merlo (2002).

[0117] Regardless of the transformation technique used, the gene is preferably incorporated into a gene transfer vector suitable for expressing Bt insecticidal toxin genes and variants in plant cells by incorporating a plant promoter into the gene transfer vector. In addition to plant promoters, promoters from a variety of sources can be used to efficiently express exogenous genes in plant cells. For example, promoters from bacteria, such as the octopine synthase promoter, nopaline synthase promoter, and mannopine synthase promoter, and promoters from viruses, such as the 35S and 19S promoters from cauliflower mosaic virus, can be used. Plant promoters include, but are not limited to, the ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), the β-conglycinin promoter, the phaseolin promoter, the ADH (alcohol dehydrogenase) promoter, heat shock promoters, the ADF (actin depolymerizing factor) promoter, and tissue-specific promoters. Promoters may also contain certain enhancer sequence elements that can improve transcription efficiency. Typical enhancers include, but are not limited to, ADH1-intron 1 and ADH1-intron 6. Constitutive promoters can be used. Constitutive promoters instruct continuous gene expression (e.g., actin, ubiquitin, CaMV 35S) in almost all cell types and almost all time. Tissue-specific promoters are responsible for gene expression in specific cells or tissue types (e.g., leaves or seeds (e.g., zein, oleosin, napin, ACP (acyl carrier protein)), and these promoters can also be used. Promoters that are active at a certain stage of plant development, as well as promoters that are active in specific plant tissues and organs, can also be used. Examples of such promoters include, but are not limited to, root-specific, pollen-specific, embryo-specific, corn silk-specific, cotton fiber-specific, seed endosperm-specific, phloem-specific promoters, etc.

[0118] In some cases, it may be necessary to use an inducible promoter. Inducible promoters are responsible for the expression of genes in response to specific signals, such as: physical stimulation (such as heat shock genes); Light (such as RUBP carboxylase); Hormones (such as glucocorticoids); Antibiotics (such as tetracycline); Metabolites; and stress (such as drought). Other desired transcription and translation elements that work in plants can be used, such as 5' non-translated leader sequences, RNA transcription termination sequences, and polyadenylic acid addition signal sequences. Many plant-specific gene transfer vectors are known in the art.

[0119] The present invention includes plant cells that are not totipotent (non-totipotent), are not propagation materials (e.g., in some embodiments, leaf cells, and in certain embodiments, exclude seed cells), and are unable to differentiate into whole plants. The present invention includes plant cells for uses other than regeneration into whole plants. For example, the plant cells can be used to produce proteins (e.g., the DIG-465 protein of the present invention). Therefore, the plant cells of the present invention include plant cells with uses other than totipotency (i.e., some cells of the present invention cannot be regenerated into whole plants). However, some embodiments do include seed cells and plant cells that can be regenerated into whole plants.

[0120] Another method for identifying the toxins and genes of the subject invention is by using oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences can be made detectable by appropriate radioactive labels, or they can be made inherently fluorescent as described in U.S. Patent No. 6,268,132. As is well known in the art, if the probe molecule and the nucleic acid sample hybridize by forming a strong base pairing bond between the two molecules, it can be reasonably inferred that the probe and the sample have considerable sequence homology. Preferably, hybridization is performed under stringent conditions by techniques well known in the art, such as those described in Keller and Manak (1993). Detection of the probe provides a means for determining in a known manner whether hybridization has occurred. This probe analysis provides a rapid method for identifying the toxin encoding genes of the subject invention. The nucleotide segments used as probes according to the present invention can be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences can also be used as PCR primers to amplify the genes of the subject invention.

[0121] As used herein, the term "stringent conditions" or "stringent hybridization conditions" is intended to mean conditions under which a probe hybridizes (anneals) to its target sequence to a detectably greater extent than to other sequences (e.g., at least 2 times greater than background). Stringent conditions are sequence-dependent and will vary in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringent conditions can be adjusted to allow for some mismatches in the sequences, thereby detecting lower degrees of similarity (heterologous probing). Typically, probes are less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

[0122] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts), at pH 7.0 to pH 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Destabilizing agents such as formamide may also be added to achieve stringent conditions. Exemplary low stringency conditions include hybridization with a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C. and washing in 1× to 2× SSC (20× SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50° C. to 55° C. Exemplary moderate stringency conditions include hybridization in 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C and a wash in 0.5x to 1x SSC at 55°C to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 0.1x SSC at 60°C to 65°C. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, typically about 4 to about 12 hours.

[0123] Specificity often varies depending on post-hybridization washes, with the key factors being the ionic strength and temperature of the final wash solution. For DNA / DNA hybrids, the thermal melting point (Tm) is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. Tm decreases by about 1°C for every 1% mismatch; therefore, Tm, hybridization conditions, and / or wash conditions can be adjusted to facilitate annealing of sequences with the desired identity. For example, if sequences with >90% identity are sought, Tm can be reduced by 10°C. Generally, stringent conditions are selected that are about 5°C lower than the Tm of the specific sequence and its complement at defined ionic strength and pH. However, high stringency conditions can utilize hybridization and / or wash at 1°C, 2°C, 3°C, or 4°C lower than the Tm; moderate stringency conditions can utilize hybridization and / or wash at 6°C, 7°C, 8°C, 9°C, or 10°C lower than the Tm; and low stringency conditions can utilize hybridization and / or wash at 11°C, 12°C, 13°C, 14°C, 15°C, or 20°C lower than the Tm.

[0124] Tm (in ° C.) can be determined experimentally or estimated by calculation. For DNA-DNA hybrids, Tm can be estimated from the equation of Meinkoth and Wahl (1984): Tm (° C.) = 81.5° C. + 16.6 (log M) + 0.41 (% GC) - 0.61 (% formamide) - 500 / L; where M is the molar concentration of the monovalent cation, % GC is the percentage of guanosine and cytidine nucleotides in the DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.

[0125] Alternatively, Tm is described by the following formula (Beltz et al., 1983).

[0126] Tm(°C)=81.5°C+16.6(log[Na+])+0.41(%GC)-0.61(%formamide)-600 / L where [Na+] is the molar concentration of sodium ions, %GC is the percentage of guanosine and cytidine nucleotides in the DNA, %formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.

[0127] Using these equations, hybridization and wash compositions, and desired Tm, one of ordinary skill will understand that variations in the stringency of the hybridization and / or wash solutions are inherently described. If the desired degree of mismatching results in a Tm below 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. Extensive guidance on nucleic acid hybridization can be found in Tijssen (1993) and Ausubel et al. (1995). See also Sambrook et al. (1989).

[0128] Hybridization of immobilized DNA on a Southern blot with a radiolabeled gene-specific probe can be performed by standard methods (Sambrook et al., supra). Radioisotopes used to label polynucleotide probes can include 32P, 33P, 14C, or 3H. Incorporation of radioisotopes into polynucleotide probe molecules can be performed by any of several methods known to those skilled in the art of molecular biology (see, for example, Sambrook et al., supra). Generally, hybridization and subsequent washes can be performed under stringent conditions that allow detection of target sequences having homology to the claimed toxin-encoding gene. For double-stranded DNA gene probes, hybridization can be carried out overnight at 20-25°C below the Tm of the DNA composition in 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg / mL denatured DNA [20X SSPE is 3 M NaCl, 0.2 M NaHPO4, and 0.02 M EDTA (sodium salt of ethylenediaminetetraacetic acid), 100X Denhardt's solution is 20 gm / L polyvinylpyrrolidone, 20 gm / L Ficoll 400 and 20 gm / L bovine serum albumin (fraction V)].

[0129] Cleaning can usually be done as follows:

[0130] (1) Wash twice in 1X SSPE, 0.1% SDS at room temperature for 15 minutes (low stringency wash).

[0131] (2) Wash once in 0.2X SSPE, 0.1% SDS at Tm-20°C for 15 minutes (moderate stringency wash).

[0132] For oligonucleotide probes, hybridization can be carried out overnight in 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg / mL denatured DNA at 10-20°C below the Tm of the hybrid. The Tm for an oligonucleotide probe can be determined by the following formula (Suggs et al., 1981).

[0133] Tm (°C) = 2 (number of T / A base pairs) + 4 (number of G / C base pairs)

[0134] Cleaning can usually be done as follows:

[0135] (1) Wash twice in 1X SSPE, 0.1% SDS at room temperature for 15 minutes (low stringency wash).

[0136] (2) Wash once in 1X SSPE, 0.1% SDS at the hybridization temperature for 15 minutes (moderate stringency wash).

[0137] Those skilled in the art will recognize that probe molecules used for hybridization and hybrid molecules formed between probe and target molecules can be detected by means other than radioactive labeling.

[0138] Variant toxin Genes and toxins useful according to the present invention include not only the disclosed truncated sequences, but also the full-length sequences, fragments, variants, mutants, and fusion proteins of these sequences that retain the characteristic insecticidal activity of the toxins specifically exemplified herein. As used herein, the term "variant" or "variation" of a gene refers to a nucleotide sequence that encodes the same toxin or an equivalent toxin having insecticidal activity. In addition, as used herein, the term "equivalent toxin" refers to a toxin that has the same or substantially the same biological activity against the target pest as the claimed toxin. Thus, a variant or variation of a claimed toxin will have at least about 30%, preferably at least about 50%, more preferably at least about 70%, and even more preferably at least about 80% of the activity of the claimed toxin. Methods for measuring insecticidal activity are well known in the art and are exemplified herein. As used herein, "variants" are intended to include proteins or polypeptides having an amino acid sequence that is at least about 60%, 65%, preferably about 70%, 75%, more preferably about 80%, 85%, and most preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. Variants also include polypeptides encoded by nucleic acid molecules that hybridize under stringent conditions to the nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, or 39. Such variants will generally retain the desired activity. Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present invention have insecticidal activity.

[0139] Variant proteins can also be designed that differ at the primary amino acid sequence level and retain the same or similar overall essential three-dimensional structure, surface charge distribution, etc. See, e.g., U.S. Patent No. 7,058,515; Larson et al., (2002); Crameri et al., (1997); Stemmer, WPC (1994a); Stemmer, WPC (1994b); Stemmer, WPC (1995); Crameri et al. (1996a); and Crameri et al. (1996b).

[0140] Some toxins of the present invention have been specifically exemplified herein. Since these toxins are merely examples of toxins of the present invention, it should be apparent that the present invention includes variants or equivalent toxins (and nucleotide sequences encoding equivalent toxins) having the same or similar insecticidal activity as the enumerated toxins. Equivalent toxins will have amino acid homology with the exemplified toxins. Amino acid identity is generally greater than 60%, preferably greater than 75%, more preferably greater than 80%, more preferably greater than 90%, and may be greater than 95%. Amino acid homology is highest in the key regions of the toxins, which are responsible for biological activity, or participate in determining the three-dimensional configuration, which is ultimately responsible for biological activity. In this regard, if certain amino acid substitutions occur in regions that are not critical to activity, or are conservative amino acid substitutions that do not affect the three-dimensional configuration of the molecule, these amino acid substitutions are acceptable and can be expected. For example, amino acids can be classified into the following categories: non-polar, uncharged polar, alkaline, and acidic. Conservative substitutions in which amino acids belonging to a certain category are replaced by amino acids belonging to the same category fall within the scope of the present invention, as long as the substitution does not substantially change the biological activity of the compound. Table 1 provides a list of examples of amino acids belonging to each category.

[0141] Table 1

[0142] Classes and Examples of Amino Acids

[0143] Amino acid category Amino acid examples Non-polar side chain Ala,Val,Leu,Ile,Pro,Met,Phe,Trp Non-charged polar side chains Gly,Ser,Thr,Cys,Tyr,Asn,Gln Acidic side chain Asp,Glu basic side chain Lys,Arg,His β-branched side chains Thr,Val,Ile Aromatic side chains Tyr,Phe,Trp,His

[0144] In some cases, non-conservative substitutions may be made. The key factor is that these substitutions do not significantly reduce the biological activity of the toxin.

[0145] Preferred insecticidal toxin proteins of the present invention are encoded by nucleotide sequences substantially identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39. "Substantially identical" refers to amino acid or nucleotide sequences having at least about 60% or 65% sequence identity, preferably about 70% or 75% sequence identity, more preferably about 80% or 85% sequence identity, and most preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to a reference sequence analyzed by one of the alignment programs described herein using standard parameters. One skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc.

[0146] In order to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, for optimal comparison purposes, sequences are aligned. The percentage identity between two sequences is a function of the number of identical positions shared by these sequences (i.e., the number of percentage identity=identical positions / total number of positions (e.g., overlapping positions) × 100). In one embodiment, the two sequences have the same length. The percentage identity between two sequences can be determined using a technique similar to that described below, with or without allowing gaps. In calculating percentage identity, the number of exact matches will typically be calculated.

[0147] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990), as modified by Karlin and Altschul (1993). This algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990). BLAST searches can be conveniently used to identify sequences in nucleic acid or protein databases that are homologous (similar) to a search sequence. BLAST nucleotide searches (score = 100, word length = 12) can be performed using the BLASTN program to identify nucleotide sequences homologous to the nucleic acid molecules claimed herein. BLAST protein searches (score = 50, word length = 3) can be performed using the BLASTX program to identify amino acid sequences homologous to the insecticidal protein molecules claimed herein.

[0148] To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al., (1997). Alternatively, PSI-Blast can be used to perform an iterated search to detect distant relationships between molecules (Altschul et al., 1997). When utilizing BLAST, gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., BLASTX and BLASTN) can be used. See www.ncbi.nlm.nih.gov. Alignments can also be performed manually by inspection.

[0149] A non-limiting example of a mathematical algorithm for comparing sequences is the ClustalW algorithm (Thompson et al., 1994). ClustalW compares sequences and aligns entire amino acid or DNA sequences, thus providing data on the sequence conservation of entire amino acid or nucleotide sequences. The ClustalW algorithm is used in several commercial DNA / amino acid analysis software packages, such as the ALIGNX module of the Vector NTI program suite (Invitrogen, Inc., Carlsbad, CA). When aligning amino acid sequences with ALIGNX, the default settings, a gap creation penalty of 10, a gap extension penalty of 0.1, and the blosum63mt2 comparison matrix can be conveniently used. After aligning two protein sequences with ALIGNX, the percentage amino acid similarity (consistency) or identity between the two sequences can be assessed. When aligning two DNA sequences with ALIGNX, the default settings, a gap creation penalty of 15, a gap extension penalty of 6.6, and the swgapdnamt comparison matrix can be conveniently used. After aligning two DNA sequences with ALIGNX, the percentage identity between the two sequences can be assessed.

[0150] Another non-limiting example of a software program that can be used to analyze ClustalW alignments is GeneDoc TM (Developed by Karl Nicholas, http: / / iubio.bio.indiana.edu / soft / molbio / ibmpc / genedoc-readme.html). GeneDoc TM Amino acid (or DNA) similarity and identity between multiple proteins can be assessed.

[0151] Another non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller (1988). Such an algorithm is integrated into the wSTRETCHER program, which is part of the wEMBOSS sequence alignment software package (accessible at http: / / emboss.sourceforge.net / ). STRETCHER uses a modification of the classical dynamic programming algorithm using linear space to calculate the optimal global alignment of two sequences. The input is a standard alignment file. The substitution matrix, gap insertion penalty, and gap extension penalty used to calculate the alignment can be specified. When using the STRETCHER program to compare nucleotide sequences, a gap opening penalty of 16 and a gap extension penalty of 4 can be used. The scoring matrix file for comparing DNA sequences is EDNAFULL. When used to compare amino acid sequences, a gap opening penalty of 12 and a gap extension penalty of 2 can be used. The scoring matrix file for comparing protein sequences is EBLOSUM62.

[0152] Another non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Needleman and Wunsch (1970), which is incorporated into the sequence alignment software packages GAP version 10 and wNEEDLE (http: / / emboss.sourceforge.net / ). Sequence identity or similarity can be determined using GAP version 10 using the following parameters: for nucleotide sequences, a gap weight of 50 and a length weight of 3 are used, and the nwsgapdna.cmp scoring matrix is ​​used to calculate % identity and % similarity. For amino acid sequence comparisons, a GAP weight of 8 and a length weight of 2 are used, and the BLOSUM62 scoring program is used to determine % identity or % similarity. wNEEDLE reads two input sequences, finds the best alignment along their entire length (including gaps), and writes their best global sequence alignment to a file. The algorithm uses a dynamic programming approach to ensure that the alignment is optimal by exploring all possible alignments and selecting the best one. A scoring matrix is ​​read containing values ​​for each possible residue or nucleotide match. wNEEDLE finds the alignment with the highest possible score, where the score of the alignment is equal to the sum of the matches taken from the scoring matrix minus the penalties incurred for opening and extending gaps in the aligned sequences. The substitution matrix and the gap opening and extension penalties are specified by the user. When comparing amino acid sequences, the default gap opening penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 comparison matrix are used. When using wNEEDLE to compare DNA sequences, the default gap opening penalty of 10, gap extension penalty of 0.5, and the EDNAFULL comparison matrix are used.

[0153] Equivalent programs may also be used. "Equivalent program" means any sequence comparison program that, for any two sequences in question, generates an alignment with identical nucleotide residue matches and the same percent sequence identity as the corresponding alignment generated by ALIGNX, wNEEDLE, or wSTRETCHER. % Identity is the percentage of identical matches between the two sequences over the reported aligned region (including any gaps in length), while % Similarity is the percentage of matches between the two sequences over the reported aligned region (including any gaps in length).

[0154] Toxin fragments and equivalentsFragments and equivalents that retain the insecticidal activity of the exemplified toxins are intended to fall within the scope of the present invention. In addition, due to the redundancy of the genetic code, the amino acid sequences disclosed herein can be encoded by a variety of different DNA sequences. It is well within the ability of those skilled in the art to create alternative DNA sequences that encode the same or substantially the same toxins. These variant DNA sequences fall within the scope of the present invention. As used herein, a "substantially identical" sequence refers to a sequence that has amino acid substitutions, deletions, additions, or insertions that do not substantially affect the insecticidal activity. Fragments that retain insecticidal activity are also included in this definition.

[0155] Alterations can be made at the amino or carboxyl termini of the insecticidal proteins and variants of the present invention to result in polypeptides that retain biological activity. Fragments or biologically active portions include polypeptide fragments that comprise an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. Biologically active portions of delta endotoxin proteins can, for example, be polypeptides that are 10, 25, 50, 100, or more amino acids in length. Such biologically active portions can be prepared and evaluated for insecticidal activity using recombinant protein engineering techniques well known in the art. Methods for determining insecticidal activity are well known in the art. As used herein, a fragment comprises at least 8 consecutive amino acids of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40, or a variant protein.

[0156] The present invention also provides fragments with improved biological activity or pest spectrum, or the ability to control resistant insect populations. Cry proteins can be modified to produce fragments that are better at forming pores and, therefore, have better insecticidal activity. In the case of tri-domain Cry proteins, domain 1 is composed of seven α-helices involved in pore formation in the midgut of susceptible insects. Modified DIG proteins with improved activity can be designed to have N-terminal deletions in regions with putative homology to α-helix 1 and α-helix 2 of domain 1.

[0157] Active fragments of these toxins can be obtained directly using proteases. Desirable fragments of the insecticidal toxins will comprise at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, or 1200 contiguous amino acids, or up to the total number of amino acids present in the full-length insecticidal toxins of the invention (e.g., 625 amino acids for SEQ ID NO: 2, or 625 amino acids for SEQ ID NO: 4).

[0158] Core toxin and protoxin chimeras. Most Bacillus thuringiensis δ-endotoxin crystal protein molecules are composed of two functional fragments. The protease-resistant core toxin is the first segment, corresponding to the roughly first half of the protein molecule. The approximately C-terminal half of the molecule is the second segment. For the purposes of this application, this second segment will be referred to as the "protoxin segment" in this article. The protoxin segment is believed to be involved in toxin crystal formation (Arvidson et al., (1989)). The complete 130kDa toxin molecule is rapidly processed into the resistant core segment by the protease in the insect intestine. Therefore, by reducing the protease processing of the toxin molecule (Haider et al., (1986)) or by reducing the solubility of the toxin (Aronson et al., (1991)), limiting the accessibility of the core to insects, the protoxin segment can deliver partial insect specificity to the toxin.

[0159] A chimeric protein advantageously connected within the toxin domain of Cry1Fa and Cry1Ab has been reported (U.S. Patent No. 5,527,883). Other successful cases in this field have been reported in the literature. For example, the construction of a hybrid δ-endotoxin is reported in the following related art. International Patent Application Publication WO 95 / 30753 discloses the construction of a hybrid Bacillus thuringiensis δ-endotoxin produced in Pseudomonas fluorescens, wherein the non-toxic protoxin fragment of Cry1F has been replaced by the non-toxic protoxin fragment of Cry1Ac / Cry1Ab disclosed in U.S. Patent No. 5,128,130. The patent also discloses the construction of a hybrid Bacillus thuringiensis δ-endotoxin produced in Pseudomonas fluorescens, wherein a portion of the non-toxic protoxin segment of CrylAc is replaced by the non-toxic protoxin fragment of the corresponding Cry1Ab. U.S. Patent No. 5,055,294 discloses the construction of a specific hybrid delta-endotoxin between Cry1Ac (amino acid residues 1-466) and Cry1Ab (amino acid residues 466-1155) for production in Pseudomonas fluorescens. Although the above patent discloses the construction of a hybrid toxin within an active toxin fragment, there are no specific details about the insecticidal activity of the hybrid toxin. International Patent Application Publication No. WO 95 / 30752 discloses the construction of a hybrid Bacillus thuringiensis delta-endotoxin for production in Pseudomonas fluorescens, wherein the non-toxic protoxin segment of Cry1C is replaced by the non-toxic protoxin segment from Cry1Ab. The above application further discloses that the activity of the hybrid delta-endotoxin against the fall armyworm is improved compared to the activity of the parent active toxin Cry1C. International Patent Application Publication No. WO 95 / 06730 discloses the construction of a hybrid Bacillus thuringiensis delta-endotoxin composed of domains 1 and 2 of Cry1E coupled to domain 3 of the non-toxic protoxin segment of Cry1C. Insect bioassays against Manduca sexta (sensitive to Cry1C and Cry1E), Spodoptera frugiperda (sensitive to Cry1C), and Spodoptera brassicae (sensitive to Cry1C) showed that the hybrid Cry1E / Cry1C hybrid toxin was active against Manduca sexta, Spodoptera frugiperda, and Spodoptera brassicae. Bioassay results are expressed as EC 50 value (toxin concentration that provides 50% growth reduction) rather than LC 50 The value (the concentration of toxin that provides 50% mortality) was obtained. Although the δ-endotoxin used in the bioassay was produced in Bacillus thuringiensis, only the active segment of the artificially produced δ-endotoxin was used, rather than the naturally occurring crystals produced by Bacillus thuringiensis that are typically present in commercial preparations of Bacillus thuringiensis. The bioassay results showed that the hybrid Cry1E / Cry1C crystals had an LC activity against Spodoptera frugiperda. 50The values ​​were 1.5 to 1.7 times lower than those of native Cry1C (i.e., more active than the latter). The art also discloses the construction of a hybrid Bacillus thuringiensis delta-endotoxin between Cry1Ab (domains 1 and 2) and Cry1C (domain 3 and a non-toxic protoxin segment), although no data on the activity or usefulness of the hybrid toxin were given.

[0160] Lee et al. (1995) reported the construction of a hybrid Bacillus thuringiensis delta-endotoxin between Cry1Ac and Cry1Aa within the active toxin segment. The active segments of artificially produced hybrid toxins were used to detect protein interactions in brush membrane vesicles (BBMVs) of sensitive insects. The biological activity of these hybrid toxins has not been reported. Honee et al. (1991) reported the construction of a hybrid delta-endotoxin between Cry1C (domain 1) and Cry1Ab (domains 2 and 3) and a hybrid between the reverse Cry1Ab (domain 1) and Cry1C (domains 2 and 3). These hybrids did not show a significant increase in activity against sensitive insects. In addition, the Cry1C (domain 1) / Cry1Ab (domains 2 and 3) hybrid toxin was found to be highly sensitive to protease degradation. A report by Schnepf et al. (1990) disclosed the construction of a CrylAc hybrid toxin in which a small portion of domain 2 was replaced by the corresponding CrylAa region, although no significant increase in activity against susceptible insect larvae was observed.

[0161] The chimeric toxins of the subject invention comprise the full core N-terminal toxin portion of a Bt toxin, and at a point beyond the end of the toxin portion, the protein transitions to a heterologous protoxin sequence. The transition to the heterologous protoxin segment can occur roughly at the natural toxin / protoxin junction, or a portion of the natural protoxin can be retained (extending beyond the toxin portion), while the transition to the heterologous protoxin occurs downstream thereof. For example, the chimeric toxins of the subject invention can have a complete toxin portion of a modified Cry1Ca toxin, such as amino acids 1-628 of DIG-473 or DIG-465, and a heterologous protoxin segment (amino acids 629 at the C-terminus). In a preferred embodiment, the heterologous protoxin segment is partially taken from Cry1Ab.

[0162] Those skilled in the art will appreciate that various Bt toxins, even within a given class, will differ to some extent in length and in the precise location of the transition from the core toxin portion to the protoxin portion. The transition from the core toxin portion to the protoxin portion typically occurs at about 50% to about 60% of the full-length toxin. The chimeric toxins of the subject invention encompass the full range of this core N-terminal toxin portion, such as the full 628 amino acids of the IRDIG544.12 insecticidal toxin protein. SEQ ID NO: 15 discloses the 1887 nucleotide sequence of the DNA encoding DIG-465, wherein the 5'-terminal 1887 nucleotides constitute the coding region for the Cry1Ca core toxin segment having the mutation L57A (leucine replaces alanine at amino acid position 57), an embodiment of the invention. SEQ ID NO: 16 discloses the 628 amino acid sequence of the full-length DIG-465 polypeptide, wherein the N-terminal core portion of Cry1Ca has the above-mentioned amino acid substitutions. SEQ ID NO: 23 discloses an 1887-nucleotide sequence of DNA encoding DIG-473, which includes the coding region of the core toxin segment of CrylCa with the mutation F596M (phenylalanine instead of methionine at amino acid position 596), another subject of the present invention. SEQ ID NO: 24 discloses a 628-amino acid sequence of the full-length DIG-473 polypeptide, which includes the portion of CrylCa with the aforementioned amino acid substitution.

[0163] Regarding the protoxin portion, the entire range of the native CrylAb protoxin portion extends from the end of the toxin portion of the CrylAb full-length protein to the C-terminus of the molecule. Note that the last approximately 100 to 150 amino acids of the protoxin are the most critical to be included in the chimeric toxin of the present invention.

[0164] Because Cry proteins have selective insecticidal activity, most Cry proteins are active only against a limited range of target pests. Therefore, there is a need to further improve the biological activity properties of Cry proteins. Cry proteins with unique binding characteristics and modes of action are useful in strategies for expanding the range of insect pests that can be controlled or for combating the development of Bt resistance.

[0165] Domain III modification.As described herein, the insecticidal toxins of the present invention are 3-domain toxins, comprising domain I, domain II, and domain III. Domain III binds to certain types of receptor proteins and may participate in the insertion of oligomeric toxin pre-pores. Some hybrid toxins comprising domain III substitutions have been shown to have excellent toxicity to Spodoptera exigua (de Maagd et al., 1996), and there is guidance (Knight et al., 2004) on the design of Cry toxin domain swaps.

[0166] Domain I modification Numerous studies using biochemical and molecular approaches have provided information on the determinants of Cry protein binding and insertion into insect midgut membranes (reviewed in Piggot and Ellar, 2007). Domain I from Cry1A and Cry3A proteins has been investigated for its ability to insert into membranes and form pores. Alpha helices 4 and 5 of domain I play key roles in membrane insertion and pore formation (Walters et al., 1993, Gazit et al., 1998; Nunez-Valdez et al., 2001), and other helices have been proposed to contact the membrane surface like the ribs of an umbrella (Gazit et al., 1998).

[0167] α-helix 3 appears to be required for oligomer prepore formation and toxicity in some cases. Some α-helix 3 mutants can bind to the receptor but do not form oligomers and are non-toxic to Manduca sexta (Jiménez-Juarez et al., 2008). However, the proteolytically activated form of Cry3Aa1 lacks α-helices 1, 2, and 3 (Carroll et al., 1997).

[0168] α-helix 1 is removed upon receptor binding. Gomez et al. (2002) found that Cry1Ab oligomers formed upon BBMV receptor binding lacked the α-helix 1 portion of domain I. Furthermore, Soberon et al. (2007) demonstrated that N-terminal deletion mutants of Cry1Ab and Cry1Ac, which lack approximately 60 amino acids comprising α-helix 1 in the three-dimensional Cry structure, were able to assemble monomers with a molecular weight of approximately 60 kDa into prepores in the absence of cadherin binding. These N-terminal deletion mutants were reported to be active against resistant insect larvae. Furthermore, Diaz-Mendoza et al. (2007) described 43 kDa and 46 kDa Cry1Ab fragments that retained activity against the Mediterranean corn borer (Sesamia nonagrioides). These fragments were shown to include amino acid residues 116 to 423; however, the exact amino acid sequences were not elucidated, and the mechanism of activity of these proteolytic fragments is unknown. The results of Gomez et al. (2002), Soberon et al., 2007 and Diaz-Mendoza et al. (2007) are in contrast to those of Hofte et al. (1986), who reported that deletion of 36 amino acids from the N-terminus of Cry1Ab resulted in loss of insecticidal activity.

[0169] Antitoxin antibodies .Using the teachings provided herein, equivalent toxins and / or genes encoding these equivalent toxins can be derived from Bacillus thuringiensis isolates and / or DNA libraries. There are many methods for obtaining the insecticidal toxins of the present invention. For example, antibodies immunoreactive to the insecticidal toxins disclosed and claimed herein can be used to identify and isolate other toxins from protein mixtures. Specifically, antibodies can be generated against the most constant portion of the toxin and the portion that is most different from other Bt toxins. These antibodies can then be used to specifically identify equivalent toxins with characteristic activity by, for example, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) or immunoblotting (western blot). Antibodies to the toxins disclosed herein or equivalent toxins or fragments of these toxins can be easily prepared using standard procedures in the art. Genes encoding these toxins can then be obtained from the microorganisms that produce the toxins.

[0170] Once the Bt insecticidal toxin has been isolated, antibodies specific for the toxin can be produced by conventional methods well known in the art. Repeated injections of selected hosts over a period of several weeks or months can elicit an immune response and result in significant anti-Bt toxin serum titers. Preferred hosts are mammalian species, with rabbits, goats, sheep, and mice being more preferred. Blood drawn from such immunized animals can be processed by conventional methods to obtain antisera (polyclonal antibodies) reactive with the Bt insecticidal toxin. The antisera can then be affinity purified by adsorption to the toxin according to techniques known in the art. The affinity-purified antisera can be further purified by separating the immunoglobulin fraction in the antisera using methods known in the art. The resulting material will be a heterogeneous population of immunoglobulins reactive with the Bt insecticidal toxin.

[0171] Anti-Bt toxin antibodies can also be produced by preparing semisynthetic immunogens consisting of synthetic peptide fragments of Bt insecticidal toxins conjugated to immunogenic carriers. Many protocols and instruments for preparing peptide fragments are well known in the art. Many suitable immunogenic carriers, such as bovine serum albumin (BSA) or keyhole limpet hemocyanin, are also well known in the art, as are techniques for coupling immunogens to carrier proteins. Once the semisynthetic immunogen is constructed, the procedures for preparing antibodies specific for Bt insecticidal toxin fragments are the same as those used to prepare antibodies reactive with native Bt toxins.

[0172] Anti-Bt toxin monoclonal antibodies (MAbs) are readily prepared using purified Bt insecticidal toxins. Methods for producing MAbs have been practiced for over 15 years and are well known to those of ordinary skill in the art. Repeated intraperitoneal or subcutaneous injections of purified Bt insecticidal toxins in an adjuvant will elicit an immune response in most animals. Hyperimmune B lymphocytes are removed from the animal and fused with a suitable fusion partner cell line that can be cultured indefinitely. Preferred animals whose B lymphocytes may be hyperimmunized and used to produce MAbs are mammals. More preferred animals are rats and mice, with the BALB / c mouse strain being most preferred.

[0173] Many mammalian cell lines are suitable fusion partners for producing hybridomas. Many such cell lines are available from the American Type Culture Collection (ATCC, Manassas, VA) and commercial suppliers. Preferred fusion partner cell lines are derived from mouse myeloma, most preferably Friendly Myeloma-653 cell line (Ventrex, Portland, ME). Once fused, the resulting hybridomas are cultured in selective growth medium for 1 to 2 weeks. Two well-known selection systems can be used to eliminate unfused myeloma cells or fusions between myeloma cells from mixed hybridoma cultures. The choice of selection system depends on the mouse strain being immunized and the myeloma fusion partner used. The AAT selection system described by Taggart and Samloff (1983) can be used; however, the HAT (hypoxanthine, aminopterin, thymidine) selection system described by Littlefield (1964) is preferred due to its compatibility with the preferred mouse strains and fusion partners described above. The spent culture medium is then screened for secretion of immunospecific MAbs. Enzyme-linked immunosorbent assay (ELISA) procedures are most suitable for this purpose; however, radioimmunoassays adapted for large-scale screening are also acceptable. Multiple screenings can be performed, designed to successively reduce the number of cultures that may be considered irrelevant or less desirable. Cultures secreting MAbs reactive with Bt insecticidal toxins can be screened for cross-reactivity with known Bt insecticidal toxins. MAbs that preferentially bind to preferred Bt insecticidal toxins can be determined for their isotype using commercially available assays. Preferred MAbs are of the IgG class, with more preferred MAbs being of the IgG1 and IgG2a subtypes.

[0174] Hybridoma cultures secreting preferred MAbs can be subcloned multiple times to establish monoclonality and stability. Well-known methods for subcloning eukaryotic non-adherent cell cultures include limiting dilution, soft agarose, and fluorescence-activated cell sorting techniques. After each subclone, the resulting cultures are preferably re-assayed for antibody secretion and isotype to ensure that a stable culture secreting the preferred MAb has been established.

[0175] Anti-Bt toxin antibodies can be used in various methods for detecting the Bt insecticidal toxins and variants or fragments thereof claimed in the present invention. It is well known that antibodies labeled with reporter groups can be used to identify the presence of antigens in a variety of environments. For decades, antibodies labeled with radioactive isotopes have been used in radioimmunoassays to identify the presence of antigens in various biological fluids with great accuracy and sensitivity. More recently, enzyme-labeled antibodies have been used to replace radiolabeled antibodies in ELISA assays. In addition, immunoreactive antibodies against the Bt insecticidal toxins of the present invention can be bound to a fixed material such as polystyrene wells or particles and used in immunoassays to determine whether the Bt toxin is present in a test sample.

[0176] In a preferred embodiment, the insecticidal protein or variant is orally delivered via a transgenic plant comprising a nucleic acid sequence expressing the toxin of the invention. The present invention provides a method for producing an insect-resistant transgenic plant comprising introducing a nucleic acid molecule of the invention into a plant, wherein the toxin can be expressed in an effective amount in the transgenic plant to control insects. In a non-limiting example, a basic cloning strategy can be to subclone a full-length or modified Cry coding sequence (CDS) into a plant expression plasmid at the NcoI and SacI restriction sites. Using, for example The resulting plant expression cassette containing the appropriate Cry coding region under the control of plant expression elements (such as a plant expressible promoter, a 3' terminal transcription terminator, and a polyadenylation addition determinant, etc.) is subcloned into a binary vector plasmid using the technique or standard restriction enzyme fragment cloning procedures. Technology, you can use LR Clonase TM (Invitrogen) is recombined into binary plant transformation plasmid with full-length and modified gene plant expression cassette.When plasmid is present in Escherichia coli and Agrobacterium cells, it is convenient to use binary plant transformation vector with bacterial gene that gives antibiotic spectinomycin resistance.It is also convenient to use binary vector plasmid containing the plant expressible selectable marker gene that has function in desired host plant.The example of plant expressible selectable marker gene includes but not limited to the aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II) that encodes resistance to antibiotic kanamycin, neomycin and G418, and those genes of encoding tolerance to glyphosate, hygromycin, methotrexate, phosphinothricin (bialaphos), imidazolinone, sulfonylurea and triazolopyrimidine herbicide such as chlorsulfuron, bromoxynil, darabond etc.

[0177] Alternatively, the plasmid structure of a binary plant transformation vector containing a DIG-465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485, DIG-487, IRDIG544.8, IRDIG544.9, IRDIG544.11 or IRDIG544.12 gene insert can be analyzed by restriction digest fingerprinting of plasmid DNA prepared from a candidate Agrobacterium isolate by standard molecular biology methods well known to those skilled in the art of Agrobacterium manipulation.

[0178] Those skilled in the art of obtaining transformed plants by Agrobacterium-mediated transformation methods will understand that Agrobacterium strains other than Z707S may be used and that the choice of strain may depend on the identity of the host plant species to be transformed.

[0179] The following are examples illustrating procedures for practicing the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight, and all solvent mixture ratios are by volume. All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated herein by reference in their entirety, to the extent they do not conflict with the explicit teachings of this specification. Unless otherwise expressly stated or implied, the terms "one," "an," and "the" as used herein mean "at least one."

[0180] Example 1

[0181] Design of plant-optimized versions of Bt insecticidal protein coding sequences

[0182] DNA sequences with plant codon bias were designed and synthesized for the production of insecticidal proteins in transgenic monocots and dicots. A codon usage table for maize (Zea mays L.) was calculated based on 706 protein-coding sequences (CDS) obtained from sequences stored in GenBank. Codon usage tables for tobacco (Nicotiana tabacum, 1268 CDS), canola (Brassica napus, 530 CDS), cotton (Gossypium hirsutum, 197 CDS), and soybean (Glycine max, approximately 1000 CDS) were downloaded from the website http: / / www.kazusa.or.jp / codon / . A biased codon set is calculated, comprising frequently used codons shared by both the maize and dicot datasets, with appropriately weighted average relative amounts of these codons. This codon set is calculated after omitting any redundant codons whose usage is less than approximately 10% of the total codon usage for that amino acid in either plant type. To obtain a plant-optimized sequence encoding an insecticidal protein, codon substitutions are made to the insecticidal protein DNA sequence so that the overall codon composition of the resulting DNA sequence conforms to a plant-optimized codon bias table. The sequence is further refined to eliminate undesirable restriction enzyme recognition sites, potential plant intron splice sites, long stretches of A / T or C / G residues, and other motifs that may interfere with RNA stability, transcription, or translation of the coding region in plant cells. Additional changes are made to introduce desired restriction enzyme recognition sites and eliminate long internal open reading frames (frames other than +1). These changes are made under the constraint of preserving the plant-biased codon composition. To complete the design, a sequence encoding a translation stop codon for all six open reading frames was added to the 3' end of the coding region, and appropriate restriction sites were added to the 5' and 3' ends of the sequence. The designed sequence was synthesized by a commercial supplier (DNA2.0, Menlo Park, CA). Additional guidance on synthetic gene production can be found in, for example, WO 97 / 13402 and U.S. Patent No. 5,380,831.

[0183] Plant-optimized DNA sequences encoding the DIG proteins of the present invention (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40) are disclosed as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39. DNA molecules comprising the sequences disclosed in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39 were synthetically assembled by a commercial entity (DNA2.0).

[0184] Example 2

[0185] Construction of expression plasmids encoding insecticidal toxins and expression in bacterial hosts

[0186] Standard cloning methods were used to construct Pseudomonas fluorescens (Pf) expression plasmids engineered to produce DIG-465 (SEQ ID NO: 16), DIG-473 (SEQ ID NO: 24), DIG-468 (SEQ ID NO: 2), DIG-483 (SEQ ID NO: 4), DIG-462 (SEQ ID NO: 10), DIG-463 (SEQ ID NO: 12), DIG-464 (SEQ ID NO: 14), DIG-466 (SEQ ID NO: 18), DIG-467 (SEQ ID NO: 20), DIG-469 (SEQ ID NO: 22), DIG-474 (SEQ ID NO: 26), DIG-482 (SEQ ID NO: 28), DIG-485 (SEQ ID NO: 6), and DIG-487 (SEQ ID NO: 8) proteins encoded by plant-optimized coding regions. Restriction endonucleases were obtained from New England BioLabs (NEB; Ipswich, MA), and T4 DNA ligase (Invitrogen Corporation, Carlsbad, CA) was used for DNA ligation.

[0187] The basic cloning strategy involves subcloning the DIG-465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485 or DIG-487 toxin coding sequence (CDS) into restriction sites such as SpeI and XhoI of pDOW1169, thereby placing it under the expression control of the Ptac promoter and rrnBT1T2 terminator from plasmid pKK223-3 (PL Pharmacia, Milwaukee, WI). pDOW1169 is a low copy plasmid with RSF1010 replication origin, pyrF gene and ribosome binding site, followed by restriction enzyme recognition site, wherein a DNA fragment containing a protein coding region can be introduced (U.S. Patent Application No. US20080193974). The expression plasmid is transformed into DC454 (a near wild-type fluorescing Pseudomonas strain with mutation ΔpyrF and lsc::lacIQI) or its derivative by electroporation, recovered in SOC-soybean hydrolysate culture medium, and plated on selective culture medium (M9 glucose agar lacking uracil, Sambrook et al., supra). The details of microbial manipulation can be disclosed in Squires, CH et al., (2004), U.S. Patent Application No. 20060008877, U.S. Patent Application No. 20080193974, and U.S. Patent Application No. 20080058262, which are incorporated herein by reference. The bacterial strain is verified by restrictive digestion of small-plasmid DNA.

[0188] Growth and expression analysis in shake flasks. DIG-465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485, or DIG-487 toxins were produced by shake flask culture of Pseudomonas fluorescens carrying the expression constructs for characterization and insect bioassays. Seed cultures were grown in M9 medium supplemented with 1% glucose and trace elements, and 50 mL of defined minimal medium (Teknova Cat. #3D7426, Hollister, CA) containing 5% glycerol was inoculated with the seed culture. After an initial incubation of 24 hours at 30°C with shaking, expression of the insecticidal protein toxin genes driven by the Ptac promoter was induced by the addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG). The cultures were sampled at the time of induction and at different times after induction. The optical density at 600 nm (OD 600) to measure cell density. Other media suitable for growing Pseudomonas fluorescens may also be used, such as those described in Huang et al., 2007 and U.S. Patent Application 20060008877.

[0189] Cell fractionation and SDS-PAGE analysis of shake flask samples. At each sampling time, 0.5 mL aliquots were centrifuged at 14,000 x g for 5 minutes. Cell pellets were frozen at -80°C. BugBuster Master Mix ( Soluble and insoluble fractions were generated from frozen shake flask cell pellet samples (Darmstadt, Germany). Each cell pellet was resuspended in 0.5 mL of BugBuster Master Mix. TM The lysate was centrifuged at 14,000 rpm for 5 minutes and the supernatant was recovered as the soluble fraction. The precipitate (insoluble fraction) was then resuspended in an equal volume of extraction buffer (8 M urea, 0.5 M NaCl, 25 mM NaPO 4, pH 10.4).

[0190] The samples were mixed 1:1 with 2X NuPAGE Tris-glycine SDS sample buffer (Invitrogen, Carlsbad, CA) containing dithiothreitol (DTT) and boiled for 5 minutes before loading onto Novex 4-20% Tris-glycine SDS polyacrylamide gels (Invitrogen, Carlsbad, CA). Electrophoresis was performed in the recommended Tris-glycine buffer. The gel was stained with Bio-Safe Coomassie stain according to the manufacturer's protocol (Bio-Rad Inc., Hercules, CA) and imaged using a GE Typhooon series imaging system (Pittsburgh, PA).

[0191] Inclusion body preparationCry protein inclusion bodies (IBs) were prepared from cells obtained from a Pseudomonas fluorescens fermentation that produced insoluble Bt insecticidal proteins (as confirmed by SDS-PAGE and MALDI-MS (matrix-assisted laser desorption / ionization mass spectrometry)). The Pseudomonas fluorescens fermentation pellet was thawed in a 37°C water bath. The cells were resuspended to 25% w / v in lysis buffer [50 mM Tris pH 7.5, 200 mM NaCl, 20 mM EDTA disodium salt (diethylaminetetraacetic acid), 1% Triton X-100, and 5 mM dithiothreitol (DTT), with 5 mL / L of bacterial protease inhibitor cocktail (P8465 Sigma-Aldrich, St. Louis, MO) added just before use]. The cells were suspended using a handheld homogenizer at the lowest setting (Tissue Tearor, BioSpec Products, Inc., Bartlesville, OK). Lysozyme (Sigma-Aldrich L7651 25 mg, from chicken egg white) was added to the cell suspension by stirring with a metal spatula, and the suspension was incubated at room temperature for 1 hour. The suspension was cooled on ice for 15 minutes and then sonicated using a Branson Sonifier 250 (twice for 1 minute, 50% duty cycle, 30% output). Cell lysis was checked microscopically. If necessary, an additional 25 mg of lysozyme was added and the incubation and sonication were repeated. When cell lysis was confirmed microscopically, the lysate was centrifuged at 11,500 × g for 25 minutes (4°C) to form an IB precipitate, and the supernatant was discarded. The IB precipitate was suspended with 100 ml of lysis buffer, homogenized with a handheld mixer and centrifuged as above. The IB precipitate was repeatedly suspended (in 50 ml of lysis buffer), washed, homogenized, sonicated and centrifuged until the supernatant became colorless and the IB precipitate became solid and off-white. For the final wash, the IB pellet was suspended in sterile filtered (0.22 μm) distilled water containing 2 mM EDTA and centrifuged.The final pellet was suspended in sterile filtered distilled water containing 2 mM EDTA and stored in 1 mL aliquots at -80°C.

[0192] The SDS-PAGE analysis and quantification of protein in IB preparations were performed as follows: 1 mL of IB precipitate was thawed and diluted 1:20 with sterile filtered distilled water. The diluted sample was then mixed with 4X reducing sample buffer [250 mM Tris, pH 6.8, 40% glycerol (v / v), 0.4% bromophenol blue (w / v), 8% SDS (w / v) and 8% β-mercaptoethanol (v / v)] and loaded onto a 4-20% Tris-glycine, 12+2 well gel (Invitrogen) run with 1X Tris / glycine / SDS buffer (BioRad). Gel was run at 200 volts for 60 minutes, then stained with Coomassie blue (50% G-250 / 50% R-250, dissolved in 45% methanol, 10% acetic acid), and decolorized with distilled water containing 7% acetic acid, 5% methanol. The target bands were quantified by comparing the optical density values ​​of the bands with bovine serum albumin (BSA) samples run on the same gel to generate a standard curve.

[0193] Dissolution of inclusion bodies . 6 mL of inclusion body suspension containing DIG-465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485 or DIG-487 protein from the Pf clone was centrifuged at the highest setting (approximately 14,000 x g) on ​​an Eppendorf 5415C centrifuge to precipitate the inclusion bodies. In a 50 mL conical tube, the stock supernatant was removed and replaced with 25 mL of 100 mM sodium carbonate buffer (pH 11). The suspension was resuspended using a pipette and vortexed to mix thoroughly. The tube was placed on a gently shaking platform at 4°C overnight to extract the target protein. The extract was centrifuged at 30,000 × g for 30 minutes at 4°C, and the resulting supernatant was concentrated 5-fold using Amicon Ultra-15 regenerated cellulose centrifugal filter devices (30,000 molecular weight cutoff; Millipore). The sample buffer was then changed to 10 mM CAPS [3-(cyclohexylamino) 1-propanesulfonic acid] pH 10 using disposable PD-10 columns (GE Healthcare, Piscataway, NJ).

[0194] Gel electrophoresis The concentrated extract was added to a 5 mM dithiothreitol reducing agent. The samples were diluted 1:50 in sample buffer (Invitrogen) and heated at 95°C for 4 minutes in preparation for electrophoresis. Five BSA standards (0.2 to 2 μg / lane) were added simultaneously to duplicate lanes of the gel (to generate a standard curve). A voltage of 200 V was applied using MOPS SDS running buffer (Invitrogen) until the tracking dye reached the bottom of the gel. The gel was stained with 0.2% Coomassie Blue G-250 in 45% methanol, 10% acetic acid and then destained, first briefly with 45% methanol, 10% acetic acid, then with 7% acetic acid, 5% methanol until the background cleared. After destaining, the gel was scanned using a Biorad Fluor-S MultiImager. The background-subtracted stained protein band volumes were obtained using the instrument's Quantity One v.4.5.2 software, and a BSA standard curve was generated for calculation of the concentration of DIG-465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485, or DIG-487 protein in the stock solution.

[0195] The expression levels of DIG-465, DIG-473, DIG-468, DIG-483, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485 and DIG-487 when expressed in Pseudomonas fluorescens bacterial cells were compared with the expression levels of truncated Cry1Ca (DIG-462). Truncated Cry1Ca (DIG-462) was expressed at approximately 1 g / l, while DIG-473 was expressed at approximately 0.5 g / l. The expression of DIG-465 was approximately 5 times that of the truncated Cry1Ca, 4.9 g / l. These in vitro results indicate that the L57A mutation results in greater expression of the truncated Cry1Ca protein.

[0196] Example 3

[0197] Insecticidal activity of DIG protein produced in Pseudomonas fluorescens

[0198] Bt insecticidal toxins DIG-462, DIG-463, DIG-464, DIG-465, DIG-466, DIG-467, DIG-468, DIG-469, DIG-470, DIG-473 and DIG-474 have been shown to be active against Lepidoptera species, including the diamondback moth (DBM; Plutellaxylostella (Linnaeus)) and the fall armyworm (FAW, Spodoptera frugiperda (Smith)).

[0199] Sample preparation and bioassay Inclusion body preparations in 10 mM CAPS pH 10 were diluted appropriately in 10 mM CAPS, pH 10, and all bioassays included a control treatment consisting of this buffer to serve as a background check for mortality or growth inhibition.

[0200] Protein concentrations in the bioassay buffer were estimated by gel electrophoresis, and a standard curve for gel densitometry was generated using BSA, which was measured using a BioRad imaging system (Fluor-S MultiImager, Quantity One software version 4.5.2). Proteins in the gel matrix were stained with a Coomassie blue-based stain and destained before reading.

[0201] The insecticidal activity of the purified proteins was tested in bioassays using newborn lepidopteran larvae fed an artificial insect diet. DBM and FAQ larvae were hatched from eggs obtained from colonies maintained by an insectary (Benzon Research Inc., Carlisle, PA). rFAW larvae were hatched from eggs obtained from proprietary colonies (Dow AgroSciences LLC, Indianapolis, IN).

[0202] These bioassays were conducted in 128-well plastic trays (C-D International, Pitman, NJ) specifically designed for insect bioassays. Each well contained 1.0 mL of multispecies lepidopteran bait (Southland Products, Lake Village, AR). A 40 μL aliquot of protein sample was applied to the 1.5 cm 2 well of each well by pipette. 2 bait surface (26.7 μL / cm 2 Cry protein concentration was calculated as per square centimeter (cm 2 The treated trays were kept in a fume hood until the liquid on the bait surface evaporated or was absorbed into the bait.

[0203] After breaking the shell a few hours, pick up individual larvae with a moistened camel hair brush and place them on the bait for processing, one larva per hole. The holes of the infected insects are then sealed with a sticky transparent plastic sheet, which is provided with pores to allow gas exchange (CD International, Pitman, NJ). The bioassay tray was kept for 5 days under controlled environmental conditions (28°C, ~60% relative humidity, 16:8 [light: dark]), and the total number of insects, dead insects, and the weight of surviving insects exposed to each protein sample were recorded then. The percentage mortality and growth inhibition percentage of each treatment were calculated. Growth inhibition (GI) was calculated as follows:

[0204] GI=[1-(TWIT / TNIT) / (TWIBC / TNIBC)]

[0205] where TWIT is the total weight of insects in the treatment,

[0206] TNIT is the total number of insects in the treatment

[0207] TWIBC is the total weight of insects in the background check (buffer control), and

[0208] TNIBC is the total number of insects in the background check (buffer control).

[0209] In the DBM bioassay, 10 and 300 ng / cm 2 The effects of DIG-462, DIG-463, DIG-464, DIG-465, DIG-466, DIG-467, DIG-468, DIG-469, DIG-470, DIG-471, DIG-472, DIG-473, and DIG-474 on this insect were compared. Inclusion body preparations of DIG-462, DIG-465, and DIG-473 were tested at 1X and 5X dilutions. Percent mortality and growth inhibition results were compared.

[0210] For DIG-462, DIG-463, DIG-464, DIG-465, DIG-466, DIG-468, DIG-469, DIG-473 and DIG-474 treatments, at 300ng / cm 2 The mortality rate was 100% under the treatment of DIG-465 and DIG-473 (Table 2 and Table 3). 2 70-90% growth inhibition was observed at 300 ng / cm 2 The lower limit was 100% growth inhibition (Table 2).

[0211] Table 2

[0212] Bioassay test results of DIG-462, DIG-465 and DIG-473 proteins on DBM,

[0213] Measuring mortality and growth inhibition

[0214] protein mortality rate Growth inhibition DIG-462 +++ ++++ DIG-465 ++ ++++ DIG-473 +++ ++++

[0215] For mortality, ++ = 10 ng / cm 2 0-20%, 300ng / cm 2 100%; +++ = 10 ng / cm 230-60% below, 300ng / cm 2 For growth inhibition, ++++ = 10 ng / cm 2 70-90% growth inhibition at 300ng / cm 2 100% growth inhibition.

[0216] Table 3

[0217] Bioassay of protein mutants

[0218] At 10 ng / cm 2 and 300ng / cm 2 Next DBM test

[0219]

[0220] Growth inhibition of FAW larvae by Cry1Ca core toxin (DIG-462), DIG-465, and DIG-473 proteins was determined to be >40% for all treatments (Table 4). Proteins were tested at full strength and diluted 5-fold with buffer (10 mM CAPS, pH 10).

[0221] Table 4

[0222] Percent growth inhibition of FAW by DIG-462, DIG-465 and DIG-473

[0223] protein Dilution % Growth Inhibition DIG-462 1X 43 DIG-462 5X 47 DIG-465 1X 81 DIG-465 5X 58 DIG-473 1X 56 DIG-473 5X 48 buffer 1X 0

[0224] The purified proteins were evaluated for DBM activity and sensitivity to chymotrypsin digestion. An unexpected and surprising finding was that DIG-473 was resistant to chymotrypsin cleavage while having the same potency against DBM as DIG-462. This was in contrast to the CrylCa core (DIG-462) and DIG-465 proteins, both of which were sensitive to chymotrypsin cleavage in vitro (Table 5).

[0225] Table 5

[0226] Proteins active against DBM (DIG-462 as a standard)

[0227] and resistance of the protein to chymotrypsin cleavage.

[0228]

[0229]

[0230] Example 4

[0231] European corn borer (ECB), southwestern corn borer (SWCB), and southern armyworm (SAW) bioassays

[0232] The bioassay was performed in a 32-well test plate. Approximately 5 mL of a 2% aqueous agar solution was added to each well and the agar was allowed to solidify completely.

[0233] Plants were approximately 3 weeks old and tested in the T1 generation. Three replicates of T1 leaf material were completed. One leaf (1" x 0.5" rectangle) was cut and placed in a single well of a tray. Each well was infested with 10 insect larvae (usually less than 24 hours old) of ECB, Cry1Fa, rECB, or SWCB. For SAW, 5 insect larvae were infested per well. Seed-based plants from the B104 inbred line and yellow fluorescent protein (YFP)-transformed plants served as negative controls.

[0234] The infected holes were then sealed with adhesive clear plastic sheets with pores to allow gas exchange (C-D International, Pitman, NJ). The trays were placed in a Conviron incubator and maintained at 28°C (16:8 h light: dark, 60% RH) for 3 days, after which the total amount of damage to each leaf was recorded (0%, 5%, 10%, 15%, 25%, 50%, 75% damage, etc., up to 100%).

[0235] When ECB and Cry1Fa-resistant ECB (rECB) insect larvae were exposed to plants containing a truncated Cry1Ca modified protein, the feeding damage caused by the insects was reduced. When the modified Cry1Ca was tested in a feeding bioassay, in which purified full-length Cry1Ca was placed on artificial insect bait and individual insects were allowed to feed on the bait containing the toxin, the modified Cry1Ca was found to be inactive against ECB and rECB. However, when expressed in corn at concentrations >120 ng / cm 2 When expressed in plants, Cry1Ca provides unexpected protection against feeding damage caused by ECB, especially rECB.

[0236] Table 6

[0237] Bioassay results of IRDIG544.12 T1 maize fed with European corn borer (ECB) and Cry1Fa-resistant ECB (rECB)

[0238]

[0239]

[0240]

[0241]

[0242] When larvae of southwestern corn borer (SWCB) and southern armyworm (SAW) were exposed to a mixture containing a truncated Cry1Ca modified protein [protein expression range of 140-340 ng / cm 2 ] plants, the feeding damage caused by both was reduced (Table 7). The average expression was 210ng / cm 2 , with a standard deviation of 35.

[0243] Table 7

[0244] Bioassay of IRDIG544.12 T1 maize plants fed with Southwestern Corn Borer (SWCB) and Southern Armyworm (SAW)

[0245] Plant name SWCB average damage Average SAW damage 112726[1]-015.AJ001.047 2 3.0 112726[1]-015.AJ001.030 4 3.0 112726[1]-015.AJ001.019 1 1.3 112726[1]-015.AJ001.034 2 2.0 YFP negative control 98.3 94.3 YFP negative control 93.3 62.5 YFP negative control 100 27.5 YFP negative control 92.7 67.5 YFP negative control 97.7 30.0 YFP negative control 100 45.0 B104 98.3 94.3 B104 100 91.7 B104 100 97.0 B104 65 70.0 B104 100 86.7

[0246] Field trials against corn borer were conducted at two locations: one in Indiana (IN), USA, and the other in Mississippi (MS), USA. Multiple constructs and events were tested for each treatment. Cry1Ab and Cry1F served as positive controls in the ECB trial. Null plants served as negative controls.

[0247] To evaluate the efficacy of ECB, each plant received 10 second-instar ECB larvae in the leaf whorls of plants at the V6-V7 stage. At MS, leaf whorls of V9 corn were also artificially infested with second-instar larvae of the southwestern corn borer (SWCB) (22 larvae per plant). The ECB and SWCB used were obtained from Benzon Laboratories. In both ECB trials, plants were evaluated for foliar damage (Guthrie 1-9 scale) (Guthrie et al., 1960) 2 weeks after infestation, where 1 is no visible damage and 9 is extensive damage to most leaves (Table 8). In the MS SWCB trial, plants were evaluated for stem damage and live insect counts 4-5 days after leaf whorl evaluation. Data collected included the number of ducts per stem, duct length, and live larvae / pupae.

[0248] Table 8

[0249] Corn borer scoring criteria (leaf wheel damage)

[0250] Fraction standard 1 No visible leaf damage, or a small amount of punch-type damage on a few leaves 2 A few leaves have small perforation damage 3 Generalized perforation lesions on several leaves 4 Perforations and elongated lesions on several leaves 5 Elongated lesions on some leaves 6 Several leaves have elongated lesions (about 1 inch). 7 Common long lesions on half of the leaves 8 Common long lesions on 2 / 3 of the leaves 9 Long lesions on most leaves

[0251] ECB field trial. ECB leaflet damage was measured to test Cry1Ca activity, which showed significantly better leaflet protection compared to null plants. Cry1Ca event activity was not statistically equivalent to the leaflet protection provided by Cry1Ab and Cry1F.

[0252] Data generated in the MS further support the unexpectedly high level of plant protection provided by Cry1Ca. High feeding pressure was established in this study. Cry1Ca demonstrated significant control compared to leaf and stem damage on null plants. Few viable insects were found in Cry1Ca stems. Cry1Ab and Cry1F events demonstrated significant leaf and stem protection compared to null plants.

[0253] Table 9

[0254] ECB impeller data, IN (average across multiple events)

[0255] Gene event Average impeller damage rating (1-9 rating) Range of mean Cry1Ca 4 2.31B 2.00-2.65 Cry1Ab 8 1.00A 1.00 Cry1F 12 1.03A 1.00-1.20 Ineffective plants 1 4.65C

[0256] Means followed by different letters are significantly different (P ≤ 0.05)

[0257] Table 10

[0258] ECB impeller and stem data, MS (averaged across events)

[0259]

[0260] For all data columns, all genetic events were significantly different from the values ​​of null plants (P<0.05). Within each column, means followed by different letters are significantly different (P≤0.05).

[0261] In the SWCB trial, only two Bt events were tested for each strain. A high feeding pressure was established in the study. Statistically equivalent stem protection and numbers of larvae and pupae per stem were measured for the Cry1Ab, Cry1F, and Cry1Ca events.

[0262] Table 11

[0263] SWCB Impeller and Stem Data, MS

[0264]

[0265] In each column, means followed by different letters are significantly different (P≤0.05).

[0266] The active form of Cry1Ca consists of amino acids 29 to 628. The full-length (1-1164) or cleaved forms (1-628 and 29-628) are active when given to insects because they are processed to the 29-628 form.

[0267] Example 5

[0268] Field trials on corn moth

[0269] Field trials against corn moth were conducted in Fowler, IN using multiple constructs and events (SEQ ID NO: 31). Null plants served as negative controls. Each plant received five first-instar larvae in the green silk of the corn cob. CEW was obtained from Benzon Laboratory. Ten corn cobs were evaluated per plot per event to assess the extent of kernel damage on cobs infested with CEW. All transgenic events provided significantly lower kernel damage levels compared to null plants. Larval ingestion of CrylCa plants was significantly inhibited (Table 12).

[0270] Table 12

[0271] CEW pellet feeding data, IN

[0272]

[0273] Example 6

[0274] Agrobacterium transformation

[0275] Binary plant transformation and expression plasmids were constructed using standard cloning methods. Agrobacterium binary plasmids containing the cry1Ca expression cassette were engineered using Invitrogen technology (Invitrogen, Carlsbad, CA) and used for Agrobacterium-mediated plant transformation. Restriction endonucleases were obtained from New England BioLabs (NEB; Ipswich, MA), and T4 DNA ligase (Invitrogen) was used for DNA ligation. LR Gateway reactions were performed using the enzyme mix (Invitrogen). Plasmid preparation kit or Plasmid preparation was performed using the Xtra Midi Kit (both from Macherey-Nagel) according to the manufacturer's instructions. After gel separation, DNA fragments were purified using the QIAquick PCR Purification Kit or the QIAEX II Gel Extraction Kit (both from Qiagen).

[0276] DNA fragments containing nucleotide sequences encoding insecticidal proteins or fragments thereof are synthesized by commercial suppliers (e.g., DNA2.0, Menlo Park, CA) and provided as cloned fragments in standard plasmid vectors, or obtained by standard molecular biology manipulations of other constructs containing appropriate nucleotide sequences. Unique restriction sites within each gene are identified, and fragments of each gene are synthesized, each containing specific deletions or insertions. The modified Cry fragments are subcloned into appropriate restriction sites in other Cry fragments to obtain regions encoding the desired full-length protein, fusion protein, or deleted variant protein.

[0277] Prepare electrocompetent cells of Agrobacterium tumefaciens strain Z707S (streptomycin-resistant derivative of Z707; Hepburn et al., 1985) and transform using electroporation (Weigel and Glazebrook, 2002). After electroporation, 1 mL of YEP broth (gm / L: yeast extract, 10; peptone, 10; NaCl, 5) is added to an electroporation cup and the cell YEP suspension is transferred to a 15 mL culture tube and incubated for 4 hours in a water bath at 28°C under continuous stirring. The cells are inoculated on YEP+ agar (25 gm / L) containing spectinomycin (200 μg / mL) and streptomycin (250 μg / mL), and the plate is incubated for 2-4 days at 28°C. Select well-separated single colonies and streak onto fresh YEP+ agar plates containing spectinomycin and streptomycin as described above, and incubate at 28°C for 1-3 days.

[0278] Template plasmid DNA was prepared from selected Agrobacterium colonies and analyzed by PCR using vector-specific primers to verify the presence of the insecticidal protein gene insert in the binary plant transformation vector. A 4 mL aliquot of cells was centrifuged from a 15 mL overnight culture grown in YEP containing spectinomycin and streptomycin as described above and extracted using Qiagen Spin MiniPreps according to the manufacturer's instructions. Control plasmid DNA was derived from the binary vector used for electroporation of Agrobacterium. PCR reactions were performed using Taq DNA polymerase from Invitrogen at a 0.5x concentration according to the manufacturer's instructions. PCR reactions were performed in an MJ Research Peltier Thermocycler programmed with the following conditions: Step 1) 94°C for 3 minutes; Step 2) 94°C for 45 seconds; Step 3) 55°C for 30 seconds; Step 4) 72°C for 1 minute per kb of expected product length; Step 5) 29 cycles to Step 2; Step 6) 72°C for 10 minutes. The reaction was maintained at 4°C after cycling. Analyze the amplified products by agarose gel electrophoresis (eg, 0.7% to 1% agarose, w / v) and visualize by ethidium bromide staining.Select one colony whose product is identical to that of the plasmid control.

[0279] Table 13

[0280] Description of plasmids used to express DIG-465 and DIG-473 in maize.

[0281] plasmids describe pDAB115752 ZmUbi1 / DIG-465 / ZmPer5::SCBV(MAM)v2 / AAD-1v3 / ZmLip pDAB115753 ZmUbi1 / DIG-473 / ZmPer5::SCBV(MAM)v2 / AAD-1v3 / ZmLip pDAB112725 ZmUbi1 / Cry1Ca(Zm) / ZmPer5::SCBV(MAM) / AAD-1v3 / ZmLip pDAB112726 ZmUbi1 / Cry1Ca(HGC) / ZmPer5::SCBV(MAM) / AAD-1v3 / ZmLip

[0282] Example 7

[0283] Production of insecticidal proteins and variants of DIG-465 and DIG-473 B.t. in monocots

[0284] Agrobacterium-mediated transformation of maize. Seeds from High II F1 hybrid seeds (Armstrong et al., 1991) were planted in 5-gallon pots containing 95% Metro-Mix 360 soilless culture medium (Sun Gro Horticulture, Bellevue, WA) and 5% clay / loam. Plants were grown in a greenhouse using a combination of high pressure sodium and metal halide lamps under a 16:8 hour light: dark photoperiod. In order to obtain immature F2 embryos for transformation, controlled sibling pollination was performed. 8-10 days after pollination, when embryo size was 1.0 to 2.0 mm, immature embryos were isolated.

[0285] Infection and co-cultivationCorn cobs were surface-disinfected by scrubbing with liquid soap, immersing in 70% ethanol for 2 minutes, then immersing in 20% commercial bleach (0.1% sodium hypochlorite) for 30 minutes, and then rinsing with sterile water. Bacteria were grown on YEP solid medium containing 100 mg / L spectinomycin, 10 mg / L tetracycline, and 250 mg / L streptomycin at 28°C for 2-3 days. One to two loopfuls of these bacteria were transferred to 5 mL of liquid infection medium (LS Basal Medium (Linsmaier and Skoog, 1965), N6 vitamins (Chu et al., 1975), 1.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 68.5 gm / L sucrose, 36.0 gm / L glucose, 6 mM L-proline, pH 5.2) to prepare a suspension of Agrobacterium cells containing the superbinary vector. The solution is vortexed until reaching uniform suspension, and the Klett-Summerson colorimeter with a purple filter is used to adjust the concentration to a final density of 200Kylett. Immature embryos are directly separated into the microcentrifuge tube containing 2mL infection culture medium. Remove culture medium, and replace with the 1mL agrobacterium solution of 200Klett units with a density, and agrobacterium and embryo solution are incubated together at room temperature for 5 minutes, then transferred to co-cultivation culture medium (LS Basal Medium, N6 vitamins, 1.5mg / L 2,4-D, 30.0gm / L sucrose, 6mM L-proline, 0.85mg / L AgNO 3, 100 μM acetosyringone, 3.0gm / L gellan gum (PhytoTechnology Laboratories, Lenexa, KS) pH 5.8) at 25 ℃, cultured 5 days under dark conditions.

[0286] After co-cultivation, embryo is transferred to selection culture medium, obtains the isolated strain of transformation in the process of about 8 weeks afterwards.For the selection of corn tissue transformed with the super binary plasmid containing the expressible pat or bar selection marker gene of plant, with based on LS culture medium (LS basal medium, N6 vitamins, 1.5mg / L 2,4-D, 0.5gm / L MES (2-(N-morpholino) ethanesulfonic acid monohydrate; PhytoTechnologies Labr.), 30.0gm / L sucrose, 6mM L-proline, 1.0mg / L AgNO , 250mg / L cefotaxime, 2.5gm / L gellan gum, pH 5.7) use together with bialaphos (Gold BioTechnology).Embryo is transferred in the selection culture medium containing 3mg / L bialaphos, until obtain embryonic isolate.By transferring to fresh selection culture medium every 2 weeks, expand the isolate of recovery for regeneration and further analysis.

[0287] Regeneration and seed production. For regeneration, the culture was transferred to "28" induction medium (MS salts and vitamins, 30 gm / L sucrose, 5 mg / L benzylaminopurine, 0.25 mg / L 2,4-D, 3 mg / L Bialaphos, 250 mg / L cefotaxime, 2.5 gm / L gellan gum, pH 5.7) under low light conditions (14 μEm-2s-1) for 1 week, then under high light conditions (about 89 μEm-2s-1) for 1 week. The tissue was subsequently transferred to "36" regeneration medium (same as induction medium, but lacking plant growth regulators). When the length of plantlet grows to 3-5 centimetres, transfer to contain SHGA medium (Schenk and Hildebrandt salts and vitamins (1972; PhytoTechnologies Lab.), 1.0gm / L inositol, 10gm / L sucrose glass culture tube and 2.0gm / L gellan gum, pH 5.8) to allow the further growth and development of shoot and root.Plant is transplanted in the identical soil mixture as mentioned above, and is grown in greenhouse to flowering.Carry out controlled pollination of seed production.

[0288] The level of expression of DIG-465 by construct 115752 and the level of expression of DIG-473 by construct 115753 were Figure 1 Both expressed their respective proteins at similar levels, and tissue samples were obtained using a leaf punch, with levels measured in leaves of approximately 70-80 ng / cm 2 .

[0289] Extracts from corn expressing a gene encoding the full-length Cry1Ca protein (MR-1206) (mw 130kDa) were subjected to SDS-PAGE. Immunoblotting using a polyclonal antibody against Cry1Ca detected at least five protein products. The full-length (130kDa) protein encoded by the gene inserted into corn was detected. The other bands represent proteolytic products of the protein. A protein fragment consisting of the amino acid sequence 1-628 representing the core toxin has a molecular weight of 70kDa. The 68kDa band represents a protein consisting of amino acids 29-628, in which the first 28 amino acids at the N-terminus are deleted. The first three bands are functionally active against Spodoptera frugiperda and other Lepidoptera insects. The fourth band represents a cleaved protein consisting of amino acids 74-628 (mw 62kDa), and the fifth band represents a Cry1Ca protein that has been further processed into amino acids 74-596 (mw 59kDa). The 62 kDa and 59 kDa bands have no functional activity against Spodoptera frugiperda and other lepidopteran insects but represent the major protein products.

[0290] Example 8

[0291] Bioassay of genetically modified corn

[0292] The biological activity of DIG-465 and DIG-473 proteins and variants produced in plant cells has been demonstrated by methods known to those skilled in the art (see, for example, Huang et al., 2006). Efficacy can be demonstrated by feeding target insects various plant tissues or tissue fragments derived from plants producing DIG-465 or DIG-473 proteins or variants in a controlled rearing environment. Alternatively, various plant tissues can be obtained from plants producing DIG-465 or DIG-473 proteins or variants, protein extracts prepared therefrom, and the protein extracts incorporated into artificial diet bioassays as described above. It should be understood that the results of such feeding assays will be compared to similarly performed bioassays using appropriate control tissues from host plants that do not produce DIG-465 or DIG-473 proteins or variants, or compared to other control samples.

[0293] The bioactivity of various events generated in maize from construct 115752 (DIG-465) was tested to see if they could prevent leaf damage caused by feeding activity of FAW or Cry1Fa-resistant FAW (rFAW). The results showed that events expressing the DIG-465 protein exhibited less feeding damage than plants that did not express the protein, and the effect was dose-dependent, with higher expression levels of DIG-465 resulting in less feeding damage caused by FAW or rFAW, an effect that appeared to be greater for rFAW (Tables 14 and 15). Figure 2 ).

[0294] Similarly, various events generated in maize from construct 115753 (DIG-473) were tested for their bioactivity in preventing leaf damage caused by feeding activity of FAW or Cry1Fa-resistant FAW (rFAW). The results showed that events expressing the DIG-473 protein exhibited less feeding damage than plants that did not express the protein, and the effect was dose-dependent, with higher DIG-473 expression resulting in less feeding damage caused by FAW or rFAW, with the effect appearing to be greater for rFAW (Tables 14 and 15). Figure 3 ).

[0295] Table 14

[0296] FAW bioassay data when exposed to DIG-465, DIG-473, or the control group

[0297]

[0298]

[0299]

[0300]

[0301] Example 9

[0302] Production of Bt insecticidal proteins and variants in dicots

[0303] Arabidopsis transformation. Arabidopsis thaliana Col-01 was transformed using the floral dip method (Weigel and Glazebrook, 2002). A selected Agrobacterium colony was used to inoculate 1 mL to 15 mL of YEP broth containing the appropriate selection antibiotic. The culture was incubated overnight at 28°C with constant agitation at 220 rpm. Each culture was used to inoculate two 500 mL cultures of YEP broth containing the appropriate selection antibiotic, and the new cultures were incubated overnight at 28°C with constant agitation. The cells were centrifuged at approximately 8700 × g for 10 minutes at room temperature, and the resulting supernatant was discarded. The cell pellet was gently resuspended in 500 mL of infiltration medium containing 1 / 2x Murashige and Skoog salts (Sigma-Aldrich) / Gamborg's B5 vitamins (Gold BioTechnology, St. Louis, MO), 10% (w / v) sucrose, 0.044 μM benzylaminopurine (10 μL / L of a 1 mg / mL DMSO stock solution), and 300 μL / L Silwet L-77. Approximately one-month-old plants were immersed in this medium for 15 seconds, being careful to ensure that the youngest inflorescences were submerged. The plants were then laid down and covered (transparent or opaque) for 24 hours, washed with water, and erected. The plants were grown at 22°C with a 16:8 light:dark photoperiod. Seeds were harvested approximately four weeks after immersion.

[0304] Arabidopsis growth and selectionFreshly harvested T1 seeds were dried at room temperature in the presence of a desiccant for at least 7 days. The seeds were suspended in a 0.1% agar / water (Sigma-Aldrich) solution and then stratified at 4°C for 2 days. In preparation for planting, 10.5 inch x 21 inch germination trays (TO Plastics Inc., Clearwater, MN) were covered with Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, WA)) with fine vermiculite and irrigated with Hoagland's solution (Hoagland and Arnon, 1950) until moist, then drained for 24 hours. The stratified seeds were sown on the vermiculite and covered with a moisture-retaining cover (KORD Products, Bramalea, Ontario, Canada) for 7 days. Under long day conditions (16:8 light: dark photoperiod), the seedlings were grown at a temperature of 120-150 μmol / m 2 Seeds were germinated in a Conviron (model CMP4030 or CMP3244; Controlled Environments Limited, Winnipeg, Manitoba, Canada) at a constant temperature (22°C) and humidity (40-50%). Plants were initially watered with Hoagland's solution and subsequently with deionized water to keep the soil moist but not wet.

[0305] After sowing, remove the moisturizing cover within 5-6 days, and spray the plant with chemical selection agent to kill the plant that germinates from unconverted seeds. For example, if the expressible selectable marker gene of the plant provided by the binary plant transformation vector is pat or bar gene (people such as Wehrmann, 1996), then the plant that can be transformed can be selected by spraying 1000X Finale solution (5.78% glufosinate-ammonium, Farnam Companies Inc., Phoenix, AZ.). Spray twice with the interval of 5-7 days subsequently. Determine survivor (actively growing plant) after final spraying 7-10 days, and be transplanted in the basin that SunshineMix LP5 is prepared. The plant of transplanting is covered 3-4 days with the moisturizing cover, and is placed in Conviron incubator under the above-mentioned growth conditions.

[0306] Those skilled in the art of dicot transformation will appreciate that other methods of selecting transformed plants are available when other plant-expressible selectable marker genes are used (eg, herbicide tolerance genes).

[0307] Example 10

[0308] Genetically modified soybeans containing DIG protein

[0309] As is known in the art, 10-20 transgenic TO soybean plants carrying an expression vector containing a nucleic acid encoding the Cry1Ca protein are generated, including, for example, by Agrobacterium-mediated transformation. Mature soybean (Glycine max) cultivar seeds are sterilized overnight with chlorine gas for 16 hours. After chlorine sterilization, the seeds are placed in an open container and placed in a LAMINAR TM The sterilized seeds were then imbibed with sterile H2O for 16 h at 24°C in the dark using a black box.

[0310] Preparation of Split Soybean Seeds The protocol for split soybean seeds containing partial hypocotyls requires preparing the soybean seed material by cutting the soybean seed material longitudinally along the hilum of the seed using a #10 blade attached to a scalpel to separate and remove the seed coat and split the seed into two cotyledonary portions. The hypocotyl is carefully partially removed, with approximately 1 / 2–1 / 3 of the hypocotyl remaining attached to the nodal end of the cotyledons.

[0311] Inoculation: The split soybean seeds containing the embryonic axis are then immersed in a solution of an Agrobacterium tumefaciens strain (e.g., EHA101 or EHA primer 105) containing a binary plasmid containing the DIG protein for approximately 30 minutes. Before immersing the cotyledons containing the embryonic axis, the Agrobacterium tumefaciens solution is diluted to a final concentration of λ = 0.6 OD 650 .

[0312] After co-cultivation inoculation, split soybean seeds were co-cultivated with Agrobacterium tumefaciens strains in a Petri dish covered with filter paper on co-cultivation medium (Wang, Kan. Agrobacterium Protocols. 2.1. New Jersey, Humana Press, 2006. Print.) for 5 days.

[0313] After 5 days of co-cultivation for shoot induction, the split soybean seeds were washed in liquid shoot induction (SI) medium, which consisted of: B5 salts, B5 vitamins, 28 mg / L ferrous iron, 38 mg / L Na2EDTA, 30 g / L sucrose, 0.6 g / L LMES, 1.11 mg / L BAP, 100 mg / L TIMENTIN TM, 200 mg / L cefotaxime and 50 mg / L vancomycin (pH 5.7). Then, the split soybean seeds were cultured on shoot induction I (SI I) medium, which consists of: B5 salts, B5 vitamins, 7 g / L Noble agar, 28 mg / L ferrous iron (Ferrous), 38 mg / L Na2EDTA, 30 g / L sucrose, 0.6 g / L MES, 1.11 mg / L BAP, 50 mg / L TIMENTIN TM , 200 mg / L cefotaxime, 50 mg / L vancomycin (pH 5.7), with the flat side of the cotyledon facing upward and the node end of the cotyledon immersed in the culture medium. After culturing for 2 weeks, explants generated from the transformed split soybean seeds were transferred to shoot induction II (SI II) medium, which contains SI I medium and supplemented with 6 mg / L glufosinate ammonium.

[0314] After 2 weeks of culturing on SI II medium, the cotyledons were removed from the explants and a flush bud pad containing the cotyledons was excised by cutting at the base of the cotyledons. The isolated bud pad from the cotyledons was transferred to bud elongation (SE) medium. SE medium consists of MS salts, 28 mg / L ferrous iron, 38 mg / L Na2EDTA, 30 g / L sucrose, 0.6 g / L MES, 50 mg / L asparagine, 100 mg / L L-pyroglutamic acid, 0.1 mg / L IAA, 0.5 mg / L GA3, 1 mg / L zeatin riboside, and 50 mg / L TIMENTIN. TM , 200 mg / L cefotaxime, 50 mg / L vancomycin, 6 mg / L glufosinate ammonium, 7 g / L Noble agar (pH 5.7). Transfer the culture to fresh SE medium every 2 weeks. Keep the culture at 24°C in CONVIRON TM In the growth room, the light intensity is 80-90 μmol / m 2 sec.

[0315] Rooting For elongated shoots emerging from the cotyledonary bud pad, rooting was promoted by cutting the elongated shoot at the base of the cotyledonary bud pad and immersing the elongated shoot in 1 mg / L IBA (indole 3-butyric acid) for 1-3 minutes. The elongated shoot was then transferred to rooting medium (MS salts, B5 vitamins, 28 mg / L ferrous iron, 38 mg / L Na2EDTA, 20 g / L sucrose and 0.59 g / L MES, 50 mg / L asparagine, 100 mg / L L-pyroglutamic acid, 7 g / L Noble agar, pH 5.6) in a plant growth tray (Phytatray).

[0316] CONVIRON was cultured at 24℃ with 18h photoperiod. TM After 1-2 weeks in the growth chamber, the rooted shoots were transferred to the soil premix in a covered sundae cup and placed in a CONVIRON TM The cells were grown in a growth chamber (models CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg, Manitoba, Canada) under long day conditions (16 h light / 8 h dark) with a light intensity of 120–150 μmol / m 2 sec, constant temperature (22°C) and humidity (40-50%) to allow the plantlets to acclimate. The rooted plantlets were acclimatized in the sundae cups for several weeks before being transferred to the greenhouse for further acclimatization and establishment of robust transgenic soybean plants.

[0317] Developmental and morphological characteristics of the transgenic lines were compared with those of non-transformed plants. Plant root, shoot, leaf, and reproductive characteristics were compared. There were no observable differences in root length and growth pattern between the transgenic and non-transformed plants. Plant characteristics such as height, leaf number and size, flowering time, flower size, and appearance were similar. In general, no observable morphological differences were observed between the transgenic lines and those that did not express the DIG protein in vitro and in soil in the greenhouse.

[0318] Example 11

[0319] Transformation of other crop species

[0320] Cotton proteins are transformed with Bt proteins (with or without chloroplast transit peptides) to provide control of lepidopteran insects by utilizing methods known to those skilled in the art, for example, essentially the same techniques as described in Example 9 of U.S. Patent 7,838,733, or Example 12 of PCT International Patent Publication No. WO 2007 / 053482.

[0321] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations thereof will occur to those skilled in the art, and that such modifications or variations are intended to be included within the spirit and scope of this application and the scope of the appended claims. Based on the teachings provided herein, those skilled in the art can readily generate and use the various toxins and polynucleotide sequences described herein.

[0322] Example Implementation

[0323] 1. A modified Cry1Ca toxin comprising residues 2 to 68 of SEQ ID NO: 2, wherein amino acid residue 54 of SEQ ID NO: 2 is selected from Gly and Ala, amino acid residue 57 of SEQ ID NO: 2 is selected from Leu and Met, and amino acid residue 68 of SEQ ID NO: 2 is selected from Val, Phe and Ile.

[0324] 2. A modified Cry1Ca toxin comprising residues 2 to 628 of SEQ ID NO:10, wherein amino acid residue 54 of SEQ ID NO:2 is selected from Gly and Ala, amino acid residue 57 of SEQ ID NO:2 is selected from Leu and Met, amino acid residue 68 of SEQ ID NO:2 is selected from Val, Phe and Ile, amino acid residue 73 of SEQ ID NO:2 is selected from Trp, Ala and Met, amino acid residue 596 of SEQ ID NO:2 is selected from Phe, Met and Ala, and amino acid residue 620 of SEQ ID NO:2 is selected from Leu and Phe.

[0325] 3. The modified CrylCa toxin of embodiment 1, further comprising a carboxyl-terminal extension consisting of amino acid residues 629 to 1164 of SEQ ID NO: 36.

[0326] 4. The modified CrylCa toxin of embodiment 2, further comprising a carboxyl-terminal extension consisting of amino acid residues 629 to 1164 of SEQ ID NO: 36.

[0327] 5. The modified CrylCa toxin of embodiment 1, further comprising an amino-terminal extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40.

[0328] 6. The modified CrylCa toxin of embodiment 2, further comprising an amino-terminal extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40.

[0329] 7. The modified CrylCa toxin of embodiment 3, further comprising an amino-terminal extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40.

[0330] 8. The modified CrylCa toxin of embodiment 4, further comprising an amino-terminal extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40.

[0331] 9. A modified Cry1Ca toxin selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40.

[0332] 10. A nucleic acid sequence encoding the modified CrylCa toxin of embodiment 1.

[0333] 11. A nucleic acid sequence encoding the modified CrylCa toxin of embodiment 2.

[0334] 12. A nucleic acid sequence encoding the modified CrylCa toxin of embodiment 3.

[0335] 13. A nucleic acid sequence encoding the modified CrylCa toxin of embodiment 4.

[0336] 14. A nucleic acid sequence encoding the modified CrylCa toxin of embodiment 5.

[0337] 15. A nucleic acid sequence encoding the modified CrylCa toxin of embodiment 6.

[0338] 16. A nucleic acid sequence encoding the modified CrylCa toxin of embodiment 7.

[0339] 17. A nucleic acid sequence encoding the modified CrylCa toxin of embodiment 8.

[0340] 18. A nucleic acid sequence selected from the group consisting 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, and SEQ ID NO: 39.

[0341] 19. A transgenic plant, plant part or seed capable of producing one or more CrylCa toxins according to embodiments 1 to 9.

[0342] 20. The transgenic plant, plant part or seed of embodiment 19 selected from the group consisting of corn, sunflower, soybean, cotton, canola, rice, sorghum, wheat, barley, vegetables, ornamentals, peppers, sugar beet, fruit, and turf grass.

[0343] 21. The transgenic plant, plant part or seed of embodiment 19, which is selected from the group consisting of corn, soybean and cotton.

[0344] 22. The transgenic plant, plant part or seed of embodiment 19, which is maize.

[0345] 23. The transgenic plant, plant part or seed of embodiment 19, which is soybean.

[0346] 24. The transgenic plant, plant part or seed of embodiment 19, which is cotton.

[0347] 25. A method of controlling insect pests of plants comprising growing transgenic plants expressing one or more modified CrylCa toxins and allowing susceptible pests to feed on the transgenic plants.

[0348] 26. The method of embodiment 25, wherein the one or more modified CrylCa toxins comprise one or more modified CrylCa toxins of embodiments 1-9.

[0349] 27. A method of controlling insect pests of plants that have developed resistance to other Cry toxins, comprising growing transgenic plants expressing one or more modified Cry1Ca toxins and allowing sensitive pests to feed on the transgenic plants.

Claims

1. A nucleic acid comprising a heterologous promoter and the nucleic acid of SEQ ID NO:

31.

2. A method of controlling European corn borer (ECB), southwestern corn borer (SWCB), southern armyworm (SAW), or corn earworm (CEW) pests, comprising growing a transgenic plant expressing one or more nucleic acid sequences of claim 1 and allowing susceptible European corn borer (ECB), southwestern corn borer (SWCB), southern armyworm (SAW), or corn earworm (CEW) pests to feed on the transgenic plant.

3. A method for controlling European corn borer (ECB) pests that have developed resistance to other Cry toxins, comprising planting transgenic plants expressing one or more nucleic acid sequences of claim 1 and allowing sensitive pests to feed on the transgenic plants.

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