Novel Genes Encoding Insecticidal Proteins

Abstract

The present invention relates to novel gene sequences encoding insecticidal proteins produced by Bacillus thuringiensis strains. Particularly, new chimeric genes encoding a Cry1C, Cry1B or Cry1D protein are provided which are useful to protect plants from insect damage. Also included herein are plant cells or plants comprising such genes and methods of making or using them, as well as plant cells or plants comprising one of such chimeric gene and at least one other of such chimeric genes.

Description

[0001] The present invention relates to new gene sequences encoding insecticidal proteins produced by Bacillus thuringiensis strains. Particularly, new chimeric genes encoding a Cry1C protein are provided which are useful to protect plants from insect damage. Also included herein are plant cells or plants comprising such genes and methods of making or using them, as well as plant cells or plants comprising such cry1C chimeric gene and at least one other gene encoding an insecticidal protein, such as new gene sequences encoding a Cry1B or Cry1D protein.

BACKGROUND OF THE INVENTION

[0002] Strain and proteins derived from Bacillus thuringiensis (abbreviated herein as "Bt") are well known for their specific toxicity to insect pests, and they have been used since almost a century to control Insect pests. Some transgenic plant species expressing Bt proteins are now available, and they successfully limit insect damage on plants. Despite the isolation of quite a number of insecticidal Bt proteins, only a few Bt proteins have been expressed in transgenic plants that have been commercialized, and this only in some crops. Most commercialized transgenic Bt plants belong to the bigger field crops such as corn and cotton. In smaller market crops such as vegetables, only a few plant species have been transformed with Bt genes so as to render them resistant to major Lepidopteran insect pests, but to date no Lepidopteran-resistant vegetable Bt-plant or seed is deregulated and marketed. Zhao et al. (2003) have described transgenic broccoli plants expressing a Cry1Ac or a Cry1C Bt toxin, as well as crosses between these plants so that both the Cry1Ac and Cry1C toxins are expressed in the same plants, but these plants have not been commercialized. NewLeaf.TM. potatoes comprising a Cry3A Coleopteran-active gene were briefly commercialized in Northern America, but have been withdrawn from the market in 2001.

[0003] The current invention provides new genes encoding proteins of the Cry1C type of Bt proteins, which ideally are combined with genes encoding proteins of the Cry1B or Cry1D type Bt proteins.

[0004] The DNA sequences of the cry1C, cry1B or cry1D genes of the invention and of the modified transit peptide of the invention (shown in the enclosed sequence listing) are artificial genes, not found in nature, and are different from any known DNA sequence. Indeed, any one of the DNA sequences of SEQ ID Nos. 1, 3, 10, 14 or 16 shows at most 76.6% sequence identity with the closest known DNA sequences.

OBJECTS AND SUMMARY OF THE INVENTION

[0005] In the current invention, several new insect control genes derived from Bt are provided for use in plants. Specifically, such genes are useful in vegetables plant crops, particularly Brassicaceae plants such as cauliflower, cabbage, Chinese cabbage, turnip, mustard, oilseed rape, kale, broccoli, Brussels sprouts, mustard spinach, and the like. Particularly, in one embodiment of this invention the following Brassica species plants are protected from insects by the new genes of the current invention: B. carinata, B. elongata, B. fruticulosa, B. juncea, B. napus, B. narinosa, B. nigra, B. oleracea, B. perviridis, B. rapa, B. rupestris, B. septiceps, B. tournefortii, and the like, particularly plants of the species Brassica oleraceae or Brassica napus. The plants or seeds comprising at least one of the new genes of the invention can be obtained by transformation of plant cells and production of plants or seed therefrom comprising the genes of the invention. Also included herein are plants or seeds obtained by crossing with a plant transformed to contain at least one of the genes of the invention, and by application of routine breeding steps. Obviously, any plant species to be protected from insect species that are killed or controlled by the Bt proteins encoded by the novel genes of this invention can be transformed with the genes of the invention to obtain transgenic plants and seeds with increased resistance to such insects.

[0006] In one embodiment, the current invention also provides a combination of technologies to allow for the most optimal product from a resistance management point of view. Indeed, in one embodiment of this invention the plants of the invention produce at least 2 different Bt proteins and such proteins are encoded by the highly-expressed cry genes of the invention which have been stably integrated, preferably at a single locus in the plant's genome. In one embodiment of the invention, such at least 2 Bt genes include a cry1C and a cry1B gene, a cry1C and a cry1D gene, or a combination of a cry1C, a cry1B and a cry1D gene of this invention. In one embodiment of the invention a marker gene allowing rapid identification of transgenic plants, preferably a herbicide resistance gene, is located in the same plant, particularly at the same locus in the plant's genome as a cry gene of the invention. In one embodiment of this invention, the marker gene is a gene encoding a phosphinothricin acetyltransferase or a glyphosate-insensitive EPSPS.

[0007] In the invention also novel cry1B and cry1D genes, particularly cry1B or cry1D chimeric genes, are provided, which can be expressed in plants at high levels, such as the cry1B1 and cry1B2 and the cry1D1 and cry1D2 genes. Also plants cells, plants or seeds comprising any of these genes and methods of producing or using them alone or in combination are provided herein.

[0008] Also, the current invention provides novel genes encoding an insecticidal protein comprising a functional plant intron in their coding sequence. The presence of the intron also secures that the gene does not express a functional protein when the gene is in an environment where the intron cannot be spliced, such as a bacteria or another prokaryotic microorganism. The presence of this intron in the gene sequence also allows for high expression levels to be obtained in plants.

[0009] Also included herein are variants of the Cry1C protein of the invention comprising the sequence of SEQ ID No. 2 from amino acid position 29 to amino acid position 627, but wherein one, some or all of the following amino acids at the following positions compared to the positions in SEQ ID No. 2 are changed: the amino acid at position 125 is Alanine, the amino acid at position 184 is Valine, the amino acid at position 295 is Arginine, the amino acid at position 454 is Aspartic acid, or the amino acid at position 593 is Arginine. Also provided herein are variants of the Cry1B protein of the invention comprising the sequence of SEQ ID No. 11 from amino acid position 31 to 648, but wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine or the amino acid at position 353 in SEQ ID No. 11 is Arginine, or a protein wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine and the amino acid at position 353 in SEQ ID No. 11 is Arginine.

[0010] Also included in this invention is a novel DNA encoding a chloroplast transit peptide, particularly a DNA comprising the sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position 371, particularly the sequence of SEQ ID No. 16, as well as such DNA encoding a variant of the protein of SEQ ID No.17, such as a chloroplast transit peptide comprising the sequence of SEQ ID No. 17 from amino acid position 3 to amino acid position 124, wherein the Cys amino acid at position 55 is replaced by Tyr and/or wherein a Gly amino acid is added after the Gly amino acid at position 51.

[0011] Specifically, the current invention provides a chimeric gene, comprising the following operably-linked sequences: a) a coding region encoding a Cry1C protein, comprising the DNA of any one of SEQ ID Nos. 1, 3, 4 or 6 or a variant thereof, and b) a promoter region capable of directing expression in plant cells. In one embodiment, such promoter comprises the sequence of SEQ ID No, 18 or 19. In another embodiment, the chimeric gene further comprises a 3' polyadenylation and transcript termination region, particularly that of the NADP-malic enzyme gene from Flaveria bidentis. In another embodiment, the chimeric gene further comprises the leader sequence of the tapetum specific E1 gene of Oryza sativa between the promoter and the coding region.

[0012] The current invention also provides a DNA comprising any of the above chimeric genes, further comprising a second chimeric gene, said second chimeric gene comprising the following operably-linked sequences: a) a second coding region encoding a Cry1B protein comprising the DNA of SEQ ID No. 8 or 10, and b) a second promoter region capable of directing expression in plant cells; or a DNA comprising any of the above chimeric genes, further comprising a second chimeric gene, said second chimeric gene comprising the following operably-linked sequences: a) a coding region encoding a Cry1D protein comprising the DNA of SEQ ID No. 12 or 14, and b) a promoter region capable of directing expression in plant cells. In one embodiment, the above DNAs are provided, wherein said second promoter region comprises the sequence of SEQ ID No. 18 or 19 and is different from said first promoter region; or wherein said second chimeric gene further comprises a 3' polyadenylation and transcript termination region, particularly of the NADP-malic enzyme gene from Flaverie bidentis. In one embodiment, the second chimeric gene in these DNAs further comprises the leader sequence of the tapetum specific E1 gene of Oryza sativa between the promoter and the coding region.

[0013] The current invention also provides the above DNAs, further comprising a third chimeric gene, said third chimeric gene comprising the following operably-linked sequences: a) a coding region encoding a Cry1D protein comprising the DNA of SEQ ID No. 12 or 14, and

[0014] b) a promoter region capable of directing expression in plants.

[0015] Also included in the current invention are a transgenic plant cell or plant, comprising any of the above genes or DNAs stably incorporated in its genome, preferably when the cell or plant is a Brassica species plant or plant cell, particularly of the species Brassica oleraceae, more particularly cabbage or cauliflower.

[0016] Also included in this invention is the use of any of the above chimeric genes or DNAs to control insect pests, to obtain plant cells, plants or seeds with increased resistance to insects; the use of any of the above chimeric genes or DNAs to delay or prevent insect resistance development in transgenic plants expressing an insecticidal protein by insects attempting to feed on such plants; or the use of any of the above chimeric genes or DNAs to obtain cabbage, oilseed rape or cauliflower protected from Plutella xylostella. Also included herein are methods for controlling insects, comprising the step of planting or sowing in a field, plants comprising any of the above chimeric genes or DNAs; as well as methods of controlling insects in Brassica species plants, comprising the step of expressing any of the above chimeric genes or DNA in plants; or methods of producing plants or seeds resistant to insects, comprising the steps of: a) obtaining a plant transformed with the gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12, and b) selecting progeny of said plant or seeds thereof, containing said gene or DNA.

[0017] Also provided in accordance with this invention is a chimeric gene comprising the following operably-linked sequences: a) a first fragment of a coding sequence encoding an insecticidal protein, b) a plant intron sequence, c) a second fragment of said coding sequence, d) a promoter region capable of directing expression in plant cells, and wherein no insecticidal protein can be produced from such chimeric gene in a given host cell wherein the intron is not spliced; particularly such chimeric gene wherein such intron is the second intron of the ST-LS1 gene of Solanum tuberosum.

[0018] Further provided herein is also a microorganism comprising any of the above chimeric genes or DNAs, particularly when such microorganism is of the genus Escherichia, Bacillus or Agrobacterium.

DESCRIPTION

[0019] In accordance with this invention, a "nucleic acid sequence" refers to a DNA or RNA molecule in single or double stranded form, preferably a DNA molecule. An "isolated DNA", as used herein, refers to a DNA which is not naturally-occurring or no longer in the natural environment wherein it was originally present, e.g., a DNA coding sequence associated with other regulatory elements in a chimeric gene, a DNA transferred into another host cell, such as a plant cell, or an artificial, synthetically-made DNA sequence having a different nucleotide sequence compared to any naturally-occurring DNA sequence.

[0020] In accordance with this invention, nucleic acid sequences, particularly DNA sequences, encoding Bt Cry toxins or variants thereof have been constructed. The new DNA sequences are designated herein as cry1C1-4, cry1B1, cry1B2, cry1D1, and cry1D2, and their encoded proteins are designated herein as Cry1C (e.g., Cry1C1, Cry1C3, and Cry1C4), Cry1B (e.g., Cry1B1 and Cry1B2) and Cry1D (e.g., Cry1D1 and Cry1D2) proteins. Also a new DNA sequence encoding a modified chloroplast transit peptide is provided herein, e.g., a DNA comprising the sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position 371, particularly the sequence of SEQ ID No. 16, which is designed for optimal expression in plants, particularly vegetables such as Brassicaceae plants, especially cabbage and cauliflower.

[0021] In accordance with this invention "Cry1C protein" refers to any insecticidal protein comprising the smallest fragment of the amino acid sequence of SEQ ID No. 2 which retains insecticidal activity (hereinafter referred to as "smallest toxic fragment"), particularly any protein comprising the amino acid sequence from the amino acid at position 29 to the amino acid at position 627 in SEQ ID No. 2, preferably any insecticidal protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 3 to amino acid position 627. Also included herein is an insecticidal protein comprising the amino acid sequence of SEQ ID No. 2 (also named Cry1C1 protein herein), SEQ ID No. 5 (also named Cry1C3 protein herein) or SEQ ID No. 7 (also named Cry1C4 protein herein).

[0022] A Cry1C protein comprising the amino acid sequence from the amino acid at position 29 to the amino acid at position 627 in SEQ ID No. 2 retains all or most of the insecticidal activity of the entire protein as produced in nature, and addition of protein sequences at the N- or C-terminal part thereof do not disrupt this activity. Hence, any protein characterized by an amino acid sequence containing or including this region is useful and forms part of this invention. This includes insecticidal hybrid or chimeric proteins comprising the smallest toxic protein fragment of the protein of SEQ ID No. 2. Also included in this definition are variants of proteins comprising the amino acid sequence from the amino acid at position 29 to the amino acid at position 627 in SEQ ID No. 2, such as insecticidal proteins comprising a sequence having a sequence identity of at least 95%, particularly at least 96%, 97%, 98% or 99% at the amino acid sequence level with this region of SEQ ID No.2, as determined using the Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) to find optimum alignment over the entire length of the sequences, using default settings (gap opening penalty 10, gap extension penalty 0.5; for amino acid sequence comparisons, the EBLOSUM62 matrix is used), preferably proteins having some, preferably 5-10, particularly less than 5, amino acids added, replaced or deleted without significantly changing, preferably without changing, the insecticidal activity of the protein. Preferred variants of the Cry1C protein of the invention include a protein comprising the sequence of SEQ ID No. 2 from amino acid position 29 to amino acid position 627, but wherein one, some or all of the following amino acids at the following positions compared to the positions in SEQ ID No. 2 are changed: the amino acid at position 125 is Alanine, the amino acid at position 184 is Valine, the amino acid at position 295 is Arginine, the amino acid at position 454 is Aspartic acid, or the amino acid at position 593 is Arginine. Also included herein are any Cry1C-based protein variants, hybrids or mutants retaining substantially the same insecticidal activity as that of the Cry1C protein of the invention defined above.

[0023] The terminology DNA or protein "comprising" a certain sequence X, as used herein, refers to a DNA or protein including or containing at least the sequence X, so that other nucleotide or amino acid sequences can be included at the 5' (or N-terminal) and/or 3' (or C-terminal) end, e.g. (the nucleotide sequence of) a selectable marker protein as disclosed in EP 0 193 259, (the nucleotide sequence of) a transit peptide, and/or a 5' or 3' leader sequence.

[0024] For the purpose of this invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (.times.100) divided by the number of positions compared. A gap, i.e., a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch 1970) in EMBOSS (Rice et al., 2000) to find optimum alignment over the entire length of the sequences, using default settings (gap opening penalty 10, gap extension penalty 0.5).

[0025] The "smallest toxic fragment" of a Cry protein of the invention, as used herein, is that smallest fragment or portion of a Cry protein retaining insecticidal activity that can be obtained by enzymatic, such as trypsin or chymotrypsin, digestion of the full length Cry protein, or that smallest fragment or portion of a Cry protein retaining insecticidal activity that can be obtained by making nucleotide deletions in the DNA encoding a Cry protein. Such smallest toxic fragment can also be obtained by treatment of a Cry protein with insect gut juice, preferably midgut juice, from an insect species susceptible to (i.e., killed or otherwise negative affected in its growth or feeding by) such Cry protein.

[0026] In accordance with this invention, "Cry1D protein" refers to any insecticidal protein comprising the smallest toxic fragment of the amino acid sequence of SEQ ID No. 15, particularly any insecticidal protein comprising the amino acid sequence from the amino acid at position 21 or 29 to the amino acid at position 604 in SEQ ID No. 15, preferably any insecticidal protein comprising the amino acid sequence of SEQ ID No. 15 from amino acid position 3 to amino acid position 604. Also included herein is an insecticidal protein comprising the amino acid sequence of SEQ ID No. 13 (also named Cry1D1 protein herein) or SEQ ID No. 15 (also named Cry1D2 protein herein). A Cry1D protein comprising the amino acid sequence from the amino acid at position 29 to the amino acid at position 604 in SEQ ID No. 15 retains all or most of the insecticidal activity of the entire protein as produced in nature, and addition of protein sequences at the N- or C-terminal part thereof do not disrupt this activity. Hence, any protein characterized by an amino acid sequence containing or including this region is useful and forms part of this invention. This includes insecticidal hybrid or chimeric proteins comprising the smallest toxic protein fragment of the protein of SEQ ID No. 15. Also included in this definition are protein variants differing in the amino acid sequence from the amino acid at position 29 to the amino acid at position 604 in SEQ ID No. 15, such as proteins with a sequence identity of at least 95%, particularly at least 97%, at least 98% or at least 99% at the amino acid sequence level in this region of SEQ ID No. 15, as determined using the Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) to find optimum alignment over the entire length of the sequences, using default settings (gap opening penalty 10, gap extension penalty 0.5, for amino acid sequence comparisons, the EBLOSUM62 matrix is used), preferably proteins having some, preferably 5-10, particularly less than 5, amino acids added, replaced or deleted in the region from the amino acid at position 29 to the amino acid at position 604 in SEQ ID No. 15 without significantly changing, preferably without changing, the insecticidal activity of the protein.

[0027] In accordance with this invention, "Cry1B protein" refers to any insecticidal protein comprising the smallest toxic fragment of the amino acid sequence of SEQ ID No. 11, particularly any insecticidal protein comprising the amino acid sequence from the amino acid at position 31 to the amino acid at position 648, in SEQ ID No. 11, preferably any insecticidal protein comprising the amino acid sequence of SEQ ID No. 11 from amino acid position 3 to amino acid position 648. Also included herein is any insecticidal protein comprising the amino acid sequence of SEQ ID No. 11 or SEQ ID No. 9. A Cry1B protein comprising the amino acid sequence from the amino acid at position 31 to the amino acid at position 648 in SEQ ID No. 11 retains all or most of the insecticidal activity of the entire protein as produced in nature, and addition of protein sequences at the N- or C-terminal part thereof do not disrupt this activity. Hence, any protein characterized by an amino acid sequence containing or including this region is useful and forms part of this invention. This includes insecticidal hybrids or chimeric proteins comprising the smallest toxic protein fragment of SEQ ID No. 11. Also included in this definition are insecticidal proteins comprising variants of the amino acid sequence from the amino acid at position 31 to the amino acid at position 648 in SEQ 1D No. 11, such as insecticidal proteins having a sequence identity of at least 80%, particularly at least 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% at the amino acid sequence level in this region of SEQ ID No. 11, as determined using pairwise alignments using the Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) to find optimum alignment over the entire length of the sequences, using default settings (gap opening penalty 10, gap extension penalty 0.5, for amino acid sequence comparisons, the EBLOSUM62 matrix is used), preferably proteins having some, preferably 5-10, particularly less than 5, amino acids added, replaced or deleted in the amino acid sequence from the amino acid at position 31 to the amino acid at position 648 in SEQ ID No. 11 without significantly changing, preferably without changing, the insecticidal activity of the protein. Preferred variants of the Cry1B protein of the invention include an insecticidal protein comprising the sequence of SEQ ID No. 11 from amino acid position 31 to 648, but wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine or the amino acid at position 353 in SEQ ID No. 11 is Arginine, or a protein wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine and the amino acid at position 353 in SEQ ID No. 11 is Arginine.

[0028] As used herein, the terms DNA or gene, as in "cry1C1 DNA", refers to any DNA sequence encoding the Cry1C, Cry1B or Cry1D protein, respectively, as defined above. This includes naturally occurring, artificial or synthetic DNA sequences encoding the Cry1C, Cry1B or Cry1D proteins defined above such as any one of SEQ ID Nos. 2, 5, 7, 9, 11, 13, 15. Also included herein are DNA sequences encoding insecticidal proteins which are similar enough to any one of the DNA sequences of SEQ ID No. 1, 3, 4, 6, 8, 10, 12, or 14 so that they can (i.e., have the ability to) hybridize to these DNA sequences under stringent hybridization conditions. Stringent hybridization conditions, as used herein, refers particularly to the following conditions: immobilizing the relevant DNA sequences on a filter, and prehybridizing the filters for either 1 to 2 hours in 50% formamide, 5% SSPE, 2.times. Denhardt's reagent and 0.1% SDS at 42.degree. C., or 1 to 2 hours in 6.times. SSC, 2.times. Denhardt's reagent and 0.1% SDS at 68 .degree. C. The denatured dig- or radio-labeled probe is then added directly to the prehybridization fluid and incubation is carried out for 16 to 24 hours at the appropriate temperature mentioned above. After incubation, the filters are then washed for 30 minutes at room temperature in 2.times. SSC, 0.1% SDS, followed by 2 washes of 30 minutes each at 68.degree. C. in 0.5.times. SSC and 0.1% SDS. An autoradiograph is established by exposing the filters for 24 to 48 hours to X-ray film (Kodak XAR-2 or equivalent) at -70.degree. C. with an intensifying screen. Of course, equivalent conditions and parameters can be used in this process while still retaining the desired stringent hybridization conditions. Preferred variants of the cry1C, cry1B or cry1D DNA of this invention are a DNA encoding the insecticidal Cry1C, Cry1B or Cry1D protein variants described above.

[0029] Also included herein as a Cry1C DNA or gene as defined herein are: a) a DNA comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide position 85 to nucleotide position 2073, b) a DNA comprising the nucleotide sequence of SEQ ID No. 3 from nucleotide position 85 to nucleotide position 2073, c) a DNA comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide position 85 to nucleotide position 2073 fused to the DNA sequence of SEQ ID No. 16, d) a DNA comprising the nucleotide sequence of SEQ ID No. 4 from nucleotide position 7 to nucleotide position 2439, e) a DNA comprising the nucleotide sequence of SEQ ID No. 3 from nucleotide position 85 to nucleotide position 2073 fused to the DNA sequence of SEQ ID No. 16, or f) a DNA comprising the nucleotide sequence of SEQ ID No. 6 from nucleotide position 7 to nucleotide position 2439.

[0030] Also included herein as a Cry1D DNA or gene as defined herein are: a) a DNA comprising the nucleotide sequence of SEQ ID No. 14 from nucleotide position 85 to nucleotide position 1812, or b) a DNA comprising the nucleotide sequence of SEQ ID No. 12 from nucleotide position 7 to nucleotide position 2178.

[0031] Also included herein as a Cry1B DNA or gene as defined herein are: a) a DNA comprising the nucleotide sequence of SEQ ID No. 8 from nucleotide position 7 to nucleotide position 2310, or b) a DNA comprising the nucleotide sequence of SEQ ID No. 10 from nucleotide position 91 to nucleotide position 1944.

[0032] The DNA sequences of the cry1C, cry1B or cry1D genes of the invention (as shown in the sequence listing, without transit peptide sequence) show at most only 76.6% sequence identity with the closest previously known DNA sequences available in databases. Available sequence databases were checked for the sequences with closest sequence identity using the well-known BLAST algorithm, and then the Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) was used to find the optimum alignment between the closest sequences and the sequences of the invention (considering their entire length, using default settings (gap opening penalty 10, gap extension penalty 0.5). For the Cry1D DNA, a fragment of the prior art sequence (of equal length) was selected to secure optimal alignment, but even then only 72.5% sequence identity was the closest sequence identity with any known DNA sequence listed in the available databases.

[0033] Hence, also included herein as cry1C, cry1B or cry1D genes are DNA sequences encoding an insecticidal protein with at least 80%, 90%, preferably at least 93 to 97%, particularly at least 98% or at least 99%, sequence identity to any one of the coding sequences of SEQ ID No. 1, 3, 4, 6, 8, 10, 12, or 14 or DNA sequences encoding an insecticidal protein hybridizing to any one of SEQ ID No. 1, 3, 4, 6, 8, 10, 12, or 14 under stringent hybridization conditions, preferably hybridizing stringently to that part of the DNA sequence of any one of SEQ ID No. 1, 3, 4, 6, 8, 10, 12, or 14 which is required to encode the smallest toxic protein fragment of the proteins of this invention. The DNA sequence identities referred to herein are calculated using the Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) to find optimum alignment over the entire length of the sequences, using default settings (gap opening penalty 10, gap extension penalty 0.5; for DNA sequence comparisons, the EDNAFULL matrix is used), the stringent hybridization conditions are as defined above.

[0034] "Insecticidal activity" of a protein, as used herein, means the capacity of a protein to kill insects, inhibit their growth or cause a reduction in insect feeding when such protein is ingested by insects, preferably by expression in a recombinant host such as a plant cell. It is understood that activity to insects of one insect species, preferably the larvae thereof, is sufficient for a protein to have insecticidal activity as used herein, although often insects of different insect species are affected by the proteins of the invention. The recombinant hosts expressing at least one of the Cry1C, Cry1B or Cry1D proteins of the invention are typically developed for or targeted to a specific major insect pest species for a certain crop or region where such insect species is a pest, e.g., the diamondback moth for Brassica plant species, but other insects will often also be controlled by the recombinant hosts of the invention, such as by the transgenic plant cells or plants, e.g., the exemplified transgenic Brassica cauliflower or cabbage plant cells or plants of the invention comprising the cry1C and/or cry1B gene in accordance with the invention.

[0035] "(Insect-)controlling amounts" of or "control" by a protein, or a recombinant host expressing a protein of this invention, as used herein, refers to an amount of protein which is sufficient to limit damage to a plant by insects feeding on such plant, e.g. by killing the insects or by inhibiting the insect development, fertility or growth in such a manner that an insect species provides less damage to a plant. This does not mean that treatment of plants with chemical insecticides will no be longer necessary (e.g., to control insect species not affected by the proteins of the invention, such as (secondary) Coleopteran or Dipteran insect pests), but that treatment by chemical insecticides for the insects targeted by the proteins of the invention can be significantly reduced or avoided, while still obtaining acceptable plant performance in the field and acceptable yield.

[0036] In accordance with this invention, insects susceptible to the new Cry proteins of the invention are contacted with these proteins in insect-controlling amounts, preferably insect-killing amounts. In one embodiment of this invention, recombinant hosts of the invention, such as transgenic plant cells or plants of the invention, express a protein or a combination of proteins of the invention at high levels, such that a "high dose" level is obtained. A "high dose level", "high dose insect resistance" or "high dose" expression, as used herein when referring to a recombinant plant cell or plant, refers to a concentration of the insecticidal protein in a plant cell or plant (measured by ELISA as a percentage of the total soluble protein, which total soluble protein is measured after extraction of soluble proteins in an extraction buffer (e.g., the extraction buffer described in Jansens et al., 1997) using Bradford analysis (Bio-Rad, Richmond, Calif.; Bradford, 1976)) which kills a developmental stage of the target insect which is significantly less susceptible, preferably between 25 to 100 times less susceptible to the toxin than the first larval stage of the insect and can thus can be expected to ensure full control of the target insect. In one embodiment this refers to the obtaining of at least 97 percent, preferably at least 99 percent, most preferably 100 percent, mortality for the fourth larval instar (for insects having 5 larval instars) or the last larval instar (for insects having 4 or less larval instars) of a target insect, as measured 10 to 14 days after insect infestation of such plant cells or plant in routine insect bioassays, preferably whole plant bioassays, using suitable controls. The existence of one target insect species (i.e., an insect species, preferably the larvae thereof, which can cause significant damage to a plant species or variety, and which is typically an insect for which a transgenic Bt plant is designed and developed) for which transformed plant cells or plants according to this invention provide a "high dose" level insect resistance is sufficient for a plant to be designated as giving "high dose" expression in accordance with this invention. Preferred target insects for the proteins of this invention are economically damaging insect pests of plants.

[0037] The terms "Cry1protein/DNA" or "Cry protein/DNA of this Invention", as used herein, refer to any one of the Cry1C, Cry1B, or Cry1D proteins or any one of the cry1C, cry1B or cry1D DNA sequences as defined herein. A Cry or Cry1 protein, as used herein, can be a protein in the full length size, also named a protoxin, or can be in a truncated form as long as the insecticidal activity is retained, or can be a combination of different proteins in a hybrid or fusion protein. A "protoxin" refers to the full length insecticidal crystal protein as it is encoded by the naturally-occurring Bt DNA sequence, a "toxin" refers to an insecticidal fragment thereof, particularly the smallest toxic fragment thereof, typically in the molecular weight range of about 50-65 kD, particularly about 60 kD, as determined by SDS-PAGE electrophoresis compared to routinely-used molecular weight standards.

[0038] A "chimeric gene", as used herein, is used to refer to a gene or DNA sequence comprising at least two different DNA fragments (such as a promoter, 5' untranslated leader, coding region, intron, 3' untranslated trailer, and a 3' end transcript formation and polyadenylation region) which are not naturally associated with each other or which originate from different sources. Typically, a plant-expressible chimeric gene, as used herein, is a gene comprising a promoter region operably-linked to a synthetic, man-made coding sequence such as any of the cry1C, cry1B or cry1D genes of the invention.

[0039] The DNA sequences encoding the Cry1 proteins of the invention can be chemically synthesized using routine techniques, and can be inserted in expression vectors to produce high amounts of Cry1 proteins. The Cry1 proteins can be used to prepare specific monoclonal or polyclonal antibodies in a conventional manner (Hofte et al., 1988) to develop immuno-assays (e.g., ELISA, Western blotting, antibody-coated dip-sticks) to detect the presence of absence of these proteins in any material, such as plant material.

[0040] The tools developed to identify transgenic plant cells, plants, or plant materials such as leaves or seeds comprising any one of the cry1 genes of the invention integrated in their genome, or DNA-containing products which comprise or are derived from plant material comprising a cry1 gene of the invention are based on the specific sequence characteristics of the novel genes of the invention, such as, a specific restriction map of the genomic region comprising the introduced (foreign) cry1 gene, molecular markers or the sequence of the foreign DNA integrated in the plant's genome.

[0041] Once the sequence of a foreign DNA such as the cry1 genes of the invention is known, primers and probes can be developed which specifically recognize these sequences in the nucleic acid (DNA or RNA) of a sample by way of a molecular biological technique. For instance a PCR method can be developed to identify the genes of the invention in biological samples (such as samples of plants, plant material or products comprising plant material). Such a PCR is based on at least two specific "primers", e.g., one recognizing a sequence within the cry1 gene and the other recognizing a sequence within the associated transit peptide sequence or within the regulatory regions such as the promoter or 3' end of the chimeric gene comprising said cry1 gene of the invention, or both recognizing specifically the cry1 gene of the invention. The primers preferably have a sequence of between 15 and 35 nucleotides which under optimized PCR conditions "specifically recognize" a sequence within the cry1 chimeric gene of the invention, so that a specific fragment ("integration fragment" or discriminating amplicon) is amplified from a nucleic acid sample comprising a cry1 gene of the invention. This means that only the targeted integration fragment, and no other sequence in the plant genome or foreign DNA, is amplified under optimized PCR conditions.

[0042] PCR primers suitable for the invention are oligonucleotides ranging in length from 17 nucleotides to about 200 nucleotides, comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 consecutive nucleotides selected from the cry1C, cry1B or cry1D chimeric gene sequence as transferred to plant cells or plants of the invention.

[0043] The primers may of course be longer than the mentioned 17 consecutive nucleotides, and may, e.g., be 20, 21, 30, 35, 50, 75, 100, 150, 200 nt long or even longer. The primers may entirely consist of nucleotide sequences selected from the cry1 nucleotide sequences. However, the nucleotide sequence of the primers at their 5' end (i.e. outside of the 3'-located 17 consecutive nucleotides) is less critical. Thus, the 5' sequence of the primers may consist of a nucleotide sequence selected from the cry1 chimeric gene sequence, as appropriate, but may contain several (e.g. 1, 2, 5, 10) mismatches. The 5' sequence of the primers may even entirely consist of a nucleotide sequence unrelated to the cry1 genes of the invention, such as a nucleotide sequence representing one or more restriction enzyme recognition sites. Such unrelated sequences or flanking DNA sequences with mismatches should preferably be no longer than 100, more preferably no longer than 50 or no longer than 25 nucleotides.

[0044] Moreover, suitable primers may comprise or consist of a nucleotide sequence at their 3' end spanning the joining region between the cry1 gene of the invention and the associated transit peptide sequence or the regulatory elements in the cry1 chimeric gene integrated in the plant DNA, such as a promoter sequence, a leader sequence, a trailer sequence or a 3' transcript termination and polyadenylation sequence. It will also be immediately clear to the skilled artisan that properly selected PCR primer pairs should also not comprise sequences complementary to each other.

[0045] The term "primer" as used herein encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR. Typically, primers are oligonucleotides from 10 to 30 nucleotides, but longer sequences can be employed. Primers may be provided in double-stranded form, though the single-stranded form is preferred. Probes can be used as primers, but are designed to bind to the target DNA or RNA and need not be used in an amplification process.

[0046] The term "recognizing" as used herein when referring to specific primers, refers to the fact that the specific primers specifically hybridize to a nucleic acid sequence in the cry1 genes of the invention under a standard PCR identification protocol, whereby the specificity is determined by the presence of positive and negative controls as is well known in the art.

[0047] Also included herein is a kit to detect the cry1 genes of the invention in biological material, as well as the use of such kit to screen biological material. A "kit" as used herein refers to a set of reagents for the purpose of performing the identification of the cry1 genes of the invention in biological samples. More particularly, a preferred embodiment of the kit of the invention comprises at least one or two specific primers, as described above. Optionally, the kit can further comprise any other reagent described herein in the PCR identification protocol. Alternatively, according to another embodiment of this invention, the kit can comprise a specific probe, as described above, which specifically hybridizes with nucleic acid of biological samples to identify the presence of the cry1 genes therein. Optionally, the kit can further comprise any other reagent (such as but not limited to hybridizing buffer, label) for identification of the cry1 genes in biological samples, using the specific probe.

[0048] Standard PCR protocols are described in the art, such as in `PCR Applications Manual" (Roche Molecular Biochemicals, 2nd Edition, 1999). The optimal conditions for the PCR, including the sequence of the specific primers, is specified in a PCR identification protocol for each cry1 gene-containing plant species. It is however understood that a number of parameters in the PCR identification protocol may need to be adjusted to specific laboratory conditions, and may be modified slightly to obtain similar results. For instance, use of a different method for preparation of DNA may require adjustment of, for instance, the amount of primers, polymerase and annealing conditions used. Similarly, the selection of other primers may dictate other optimal conditions for the PCR identification protocol. These adjustments will however be apparent to a person skilled in the art, and are furthermore detailed in current PCR application manuals such as the one cited above.

[0049] Examples of suitable primer combinations in accordance with the invention are (sequence 5'-3') for the cry1B gene of the invention: P1B227 (TAC TTC GAA CAG AAA GAA CGA GAA CGA G, SEQ ID No. 20) and P1B228 (GTC CAG CGA AAG GAA CTC CAA GAA, SEQ ID No. 21), and for the cry1C gene of the invention: P1C247 (AAC CTT GAG GGA CTT GGA AAC, SEQ ID No. 22) and P1C252 (AAG ATG AGG GTT TCT GAT AGC AG, SEQ ID No. 23). Hence, any gene encoding an insecticidal Cry1B or Cry1C protein and specifically recognized by these primers is included herein, as well as any method to detect such genes using such or other specific primers.

[0050] Also specific markers or labeled probes can be designed to detect the DNA sequences of this invention, and any use of specific markers or probes directed to any of the cry1C, cry1B or cry1D genes of the invention is included herein. In one embodiment of this invention, the specific markers, primers or labeled probes do not detect or recognize any plant, preferably any plant of the same species as the test plant, not containing a cry1 DNA sequence of the invention, particularly any such markers, primers or labeled probes do not detect or recognize any plant expressing a Cry1C, Cry1D or Cry1B protein wherein such plant does not contain a DNA sequence of the invention (such as a cry1C, cry1D or cry1B DNA as defined herein, e.g., a DNA comprising the nucleotide sequence of any one of SEQ ID No. 1, 3, 4, 6, 8, 10, 12, or 14).

[0051] The DNA sequences of this invention can be slightly modified to allow for more convenient restriction enzyme sites, or to make small changes without changing the efficacy and without significantly changing, preferably without changing, the protein they encode. Indeed, because of the degeneracy of the genetic code, it is well known that most amino acid codons can be replaced by others without changing the amino acid sequence of the protein. Furthermore, some amino acids can be substituted by other equivalent amino acids without significantly changing, preferably without changing, the insecticidal activity of the protein. Also, changes in amino acid sequence or composition in regions of the molecule, different from those responsible for binding or pore formation are less likely to cause a difference in insecticidal activity of the protein (e.g., the C-terminal part of the Cry1 protoxin can be removed or be replaced by another amino acid sequence without affecting the insecticidal activity of the Cry1 proteins of the invention). Equivalents of the DNA sequences of the invention include DNA sequences with less than 20, preferably 5-10, nucleotide differences compared to the cry1 genes of this invention as defined herein, but which encode an insecticidal Cry1 protein of the invention, as defined herein.

[0052] Small modifications to a DNA sequence such as described above can be routinely made, i.e., by PCR-mediated mutagenesis (Ho et al.,1989, White et al., 1989). More profound modifications to a DNA sequence can be routinely done by de novo DNA synthesis of a desired coding region using available techniques.

[0053] The term "encoding", as used herein, when referring to a gene encoding a protein, refers to the capacity of such gene to produce a protein upon transcription and translation of the coding sequence contained in such gene in a target host cell. Hence, the cry1C1 chimeric gene of the invention encodes the Cry1C1 protein of the invention, even though this gene contains two coding sequences interrupted by a non-coding intron sequence.

[0054] With the term "substantially the same", when referring to the amino acid sequence of a Cry1 protein of this invention, is meant to include an amino acid sequence that differs in no more than 5%, preferably no more than 2%, to the amino acid sequence of the protein compared to; and when referring to toxicity of Cry protein, is meant to include a protein whose LC50 value obtained under the same conditions of bio-assay (preferably in the same bio-assay using insects from the same population and suitable controls) differs no more then 2 times, preferably no more than 50%, of the LC50 value obtained for the protein compared to.

[0055] "Microorganism", as used herein, refers to any living organism that can be observed only with the aid of a microscope, such as bacteria, yeast cells, plant cells, viruses, fungi. This includes all generally unicellular organisms with dimensions beneath the limits of vision which can be propagated and manipulated in a laboratory, typically prokaryotic or unicellular eukaryotic life forms, including tissue cultures and plasmids.

[0056] The cry1 DNA sequences of the invention, prepared from total DNA, can be ligated in suitable expression vectors and transformed in suitable host cells which can then be screened by conventional detection tools for presence and expression of the toxin.

[0057] A database search with the genes of this invention indicates that the DNA sequences of the invention are significantly different from any previously described genes or DNA sequences encoding toxins with activity against Lepidoptera (see, e.g., the Jan. 26, 2006 version of DNA sequences described in patent applications (Geneseq release 200602), Hofte and Whiteley, 1989; Crickmore et al., 1998; and the Aug. 2, 2005 update on the Bt nomenclature website corresponding to the Crickmore et al. (1998) publication, found at:

[0058] http://www.lifesci.sussex.ac.uk/home/Neil Crickmore/Bt/index.html).

[0059] The closest sequence identity at the DNA level (for the entire length of the sequences of the invention) in available DNA sequence databases (from patent or scientific literature) was 76.60% for the cry1C DNA of SEQ ID No. 1 or 3, 73% for the cry1B DNA of SEQ ID No. 10, and 72.5% for the cry1D DNA of SEQ ID No. 14, using the above defined Needleman-Wunsch default settings in EMBOSS. Hence, assuming the available DNA sequence databases are representative of all known DNA sequences, the DNA sequences of this invention differ in at least 23% of their nucleotides from any previously known DNA sequence. Assuming the closest sequences are contained in the available databases, this reflects a difference in about 485 nucleotides for the nucleotide sequence of SEQ ID No. 1 or 3, a difference in about 524 nucleotides for the nucleotide sequence of SEQ ID No. 10, and a difference in about 498 nucleotides for the nucleotide sequence of SEQ ID No. 14 with their respective closest published DNA sequence. This difference will be even more pronounced for the DNA sequences of SEQ ID No. 4, 6, 8, or 12, which encode a fusion protein with a transit peptide. Also the optimized chloroplast transit peptide DNA sequence of this invention (SEQ ID No. 16), which was adapted for expression in the target plants of the invention, was found to have about 76.1% sequence identity (for that part of equal length to the SEQ ID No. 16 sequence) to the closest DNA sequence identified in available DNA sequence databases and hence is very different.

[0060] By an "insecticidally effective part (portion or fragment)" of DNA sequences encoding a Cry1 protein, also referred to herein as "truncated gene" or "truncated DNA", is meant a DNA sequence encoding a polypeptide which has fewer amino acids than the Cry1 protein protoxin form but which is still insecticidal.

[0061] In order to express all or an insecticidally effective part of the DNA sequence encoding a Cry protein of this invention in a recombinant host such as E. coli, in other Bt strains or in plants, suitable restriction sites can be introduced, flanking the DNA sequence. This can be done by site-directed mutagenesis, using well-known procedures (Stanssens et al., 1989; White et al., 1989). In order to obtain improved expression in plants, the cry1 genes of the invention are artificial genes, wherein the sequence has been adapted for optimal expression by DNA synthesis. In such sequence, replacement of DNA sequences inhibiting optimal expression is achieved by designing DNA sequences comprising codons more preferred by plants, preferably the target plant genus or species.

[0062] For obtaining enhanced expression in plants or preventing expression of an insecticidal protein when not present in a plant host cell (such as in a bacterial host cell), in one embodiments of the invention a plant intron is inserted in the chimeric cry1 genes of the invention, preferably in the coding sequence of at least one of the cry1 genes of the invention. Any of the known plant introns (e.g., Brown, 1986, Brown and Simpson, 1998, Brown et al., 1996) can be used herein as long as it is operably-linked to the coding sequence fragments so as to assure proper splicing. Operable linkage of the intron and the resulting proper splicing is conveniently checked in the target host plant species or cells thereof by RT-PCR or Northern blot or by any other means available in the art. In one embodiment an intron of a dicot plant gene is used in genes to be expressed in dicot plant cells, and a monocot intron is used in genes to be expressed in monocot plants. In one embodiment, the intron of the invention is the second intron of the light-inducible tissue-specific ST-LS1 gene of Solanum tuberosum (potato) as described by Eckes et al. (1986), e.g., the nucleotide sequence of SEQ ID No. 1 between nucleotide position 672 and 862. In one embodiment of this invention a plant intron is introduced into any Bt insecticidal protein coding sequence, particularly the intron of SEQ ID No: 1 between nucleotide position 672 and 862, so that it is effectively spliced in plant cells. Effective splicing in plants cells can be measured using routine techniques, such as RT-PCR, Northern blotting, or the detection of a functional protein produced in plant cells. Of course, for effective splicing the intron needs to be inserted in the correct position of the coding sequence so that functional 5' and 3' splice sites are obtained in the sequence. The two cry genes of the invention, illustrated in SEQ ID Nos. 1 and 3, each containing a plant intron at a different location, were found by RT-PCR analysis to both be effectively spliced in Brassica oleraceae plant cells, and to produce an mRNA encoding the expected Cry protein.

[0063] In accordance with one embodiment of this invention, the proteins are targeted to intracellular organelles such as plastids, preferably chloroplasts, mitochondria, or are secreted from the cell, potentially optimizing protein stability and/or expression. For this purpose, the chimeric genes of the invention comprise a coding region encoding a signal or target peptide, linked to the Cry protein coding region of the invention. Particularly preferred peptides to be included in the proteins of this invention are the transit peptides for chloroplast or other plastid targeting, especially duplicated transit peptide regions from plant genes whose gene product is targeted to the plastids, the optimized transit peptide described by Lebrun et al. (1996), or Capellades et al. (U.S. Pat. No. 5,635,618), the transit peptide of ferredoxin-NADP+oxidoreductase from spinach (Oelmuller et al., 1993), the transit peptide described in Wong et al. (1992) and the targeting peptides in published PCT patent application WO 00/26371. In one embodiment of the invention, the chloroplast transit peptide comprises the sequence of SEQ ID No. 17 from amino acid position 3 to amino acid position 124 or variant thereof, such as a chloroplast transit peptide comprising the sequence of SEQ ID No. 17 from amino acid position 3 to amino acid position 124, wherein the Cys amino acid at position 55 is replaced by Tyr in SEQ ID No. 17 and/or wherein a Gly amino acid is added after the Gly amino acid at position 51 in SEQ ID No. 17. Also preferred are peptides signalling secretion of a protein linked to such peptide outside the cell, such as the secretion signal of the potato proteinase inhibitor II (Keil et al., 1986), the secretion signal of the alpha-amylase 3 gene of rice (Sutliff et al., 1991) and the secretion signal of tobacco PR1 protein (Cornelissen et al., 1986).

[0064] Particularly useful signal peptides in accordance with the invention include the chloroplast transit peptide (e.g., Van Den Broeck et al. (1985), or the optimized chloroplast transit peptide of U.S. Pat. No. 5,510,471 and U.S. Pat. No. 5,635,618 causing transport of the protein to the chloroplasts, a secretory signal peptide or a peptide targeting the protein to other plastids, mitochondria, the ER, or another organelle. Signal sequences for targeting to intracellular organelles or for secretion outside the plant cell or to the cell wall are found in naturally targeted or secreted proteins, preferably those described by Klosgen et al. (1989), Klosgen and Well (1991), Neuhaus & Rogers (1998), Bib et al. (1999), Morris et al. (1999), Hesse et al. (1989), Tavladoraki et al. (1998), Terashima et al. (1999), Park et al. (1997), Shcherban et al. (1995), all of which are incorporated herein by reference, particularly the signal peptide sequences from targeted or secreted proteins of Brassica plant species, corn, cotton, or soybean. A preferred DNA sequence encoding a transit peptide of the invention is a DNA comprising the sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position 371, particularly the sequence of SEQ ID No. 16.

[0065] Furthermore, for any target pest insect, the binding properties of the Cry proteins of the invention can be evaluated, using methods known in the art (e.g., Van Rie et al., 1990), to determine if the Cry1 proteins of the invention bind to sites on a target insect midgut that are not recognized (or competed for) by other Cry or non-Cry proteins. Other Bt toxins binding to different binding sites in relevant susceptible insects, or other toxins derived from Bt strains or other sources (such as VIP toxins or insect (gut) proteinase inhibitors) with a different mode of action are very valuable to also express in a plant in addition to any one of the cry1 genes herein, to prevent or delay the development of insect resistance to a plant expressing insecticidal toxins. Because of the characteristics of the new cry1 genes, they are extremely useful for transforming plants, e.g. monocots such as corn or wheat and dicots such as cotton, soybean and Brassica species plants, to protect these plants from insect damage.

[0066] Especially for insect resistance management purposes for a specific insect pest, it is preferred to combine a cry1C gene of this invention with another gene encoding a different insect control protein, particularly a Bt crystal protein, which does not recognize at least one binding site recognized by such Cry1C protein in a target insect. Preferred insect control proteins to combine with the Cry1C proteins of this invention, particularly for simultaneous expression in plants, preferably Brassica species plants, particularly cabbage and cauliflower, include the Cry1B protein of this invention or the Cry1D protein of this invention, the VIP3Aa protein or a toxic fragment thereof as described in Estruch et al., 1996 and U.S. Pat. No. 6,291,156, or insecticidal proteins from Xhenorhabdus, Serratia or Photorhabdus species strains (e.g., Waterfield et al., 2001; ffrench-Constant and Bowen, 2000). In one embodiment, such co-expression is obtained by transforming a plant already expressing an insect control protein with a cry1 gene of this invention, or by crossing plants transformed with the insect control protein and plants transformed with the cry1 gene of this invention. For Brassica species plants, preferably the cry1 gene is used as first gene and as second gene the Cry1B, Cry1D or VIP3Aa protein or variants or derivatives thereof are used. Methods for obtaining expression of different Bt (or similarly, for other insect control proteins) insecticidal proteins in the same plant in an effort to minimize or prevent resistance development to transgenic insect-resistant plants are described in EP patent 0 408 403. In one embodiment of the invention, the cry1C gene of the invention is located in one and the same locus as a second insect control gene, such as a Cry1B or Cry1D gene, in the transgenic plant cells or plants of the invention, so that these genes do not segregate in the progeny of such plant cells or plants.

[0067] Preferably, for selection purposes but also for increasing the weed control options, the transgenic plants of the invention are also transformed with a DNA encoding a protein inactivating a broad-spectrum herbicide or encoding a protein which is a variant of the protein target for the herbicide but which protein variant is insensitive to such herbicide, e.g., herbicides based on glufosinate or glyphosate.

[0068] The insecticidally effective cry1 gene, preferably the cry1 chimeric gene, encoding an insecticidally effective portion of the Cry protoxin, can be stably inserted in a conventional manner into the nuclear genome of a plant cell, and the so-transformed plant cell can be used in a conventional manner to produce a transformed plant that is insect-resistant. In this regard, a disarmed Ti-plasmid, containing the insecticidally effective cry1 gene part, in Agrobacterium, e.g., Agrobacterium tumefaciens can be used to transform the plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 0 116 718, EP 0 270 822, PCT publication WO 84/02913 and published European Patent application ("EP") 0 242 246 and in De Block et al. (1989). Preferred Ti-plasmid vectors each contain the insecticidally effective cry gene part between the border sequences, or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid. Of course, other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0 233 247), pollen mediated transformation (as described, for example in EP 0 270 356, PCT publication WO 85/01856, and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation (as described, for example in EP 0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (as described, for example in U.S. Pat. No. 4,536,475), and other methods such as the methods for transforming certain lines of corn (e.g., U.S. Pat. No. 6,140,553; Fromm et al., 1990; Gordon-Kamm et al., 1990) and the method for transforming monocots generally (PCT publication WO 92/09696). For cotton transformation, especially preferred is the method described in PCT patent publication WO 00/71733. For soybean transformation, reference is made to methods known in the art, e.g., Hinchee et al. (1988) and Christou et al. (1990) or the method of WO 00/42207.

[0069] Also, besides transformation of the nuclear genome, also transformation of the plastid genome, preferably chloroplast genome, is included in the invention. Kota et al. (1999) have described a method to express a Cry2A protein in tobacco chloroplasts, and Lin et al. (2003) described expression of a cry1C gene in transplastomic tobacco plants.

[0070] The resulting transformed plant can be used in a conventional plant breeding scheme to produce more transformed plants with the same characteristics or to introduce the insecticidally effective cry gene part in other varieties of the same or related plant species. Seeds, which are obtained from the transformed plants, contain the insecticidally effective cry gene part as a stable genomic insert.

[0071] The insecticidally effective cry1 gene, preferably the sequence of SEQ ID No. 1, 3, 4 or 6, is inserted in a plant cell genome so that the inserted gene is downstream (i.e., 3') of, and under the control of, a promoter which can direct expression of the gene in a plant cell (herein named a "plant-expressible promoter"). This is preferably accomplished by inserting the cry1 chimeric gene comprising a plant-expressible promoter in the plant cell genome, particularly in the nuclear or plastid (e.g., chloroplast) genome. Preferred plant-expressible promoters include: the strong constitutive 35S promoters (the "35S promoters") of the cauliflower mosaic virus (CaMV) of isolates CM 1841 (Gardner et al., 1981), CabbB-S (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); the 35S promoter described by Odell et al. (1985), promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen et al., 1992, see also Comejo et al., 1993), the gos2 promoter (de Pater et al., 1992), the emu promoter (Last et al., 1990), Arabidopsis actin promoters such as the promoter described by An et al. (1996), rice actin promoters such as the promoter described by Zhang et al. (1991); promoters of the Cassava vein mosaic virus (WO 97/48819, Verdaguer et al. (1998)), the pPLEX series of promoters from Subterranean Clover Stunt Virus (WO 96/06932), particularly the duplicated promoter region derived from the subterranean clover stunt virus genome segment 4 or 7 (referred to as the "S7S7" or "S4S4" promoters herein) described by Boevink et al. (1995) or Schunmann et al. (2003), an alcohol dehydrogenase promoter, e.g., pAdh1S (GenBank accession numbers X04049, X00581), and the TR1' promoter and the TR2' promoter (the "TR1' promoter" and "TR2' promoter", respectively) which drive the expression of the 1' and 2' genes, respectively, of the T-DNA (Velten et al., 1984). Alternatively, a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (e.g., leaves and/or roots) whereby the inserted cry gene part is expressed only in cells of the specific tissue(s) or organ(s). For example, the insecticidally effective cry gene part could be selectively expressed in the leaves of a plant (e.g., corn, cotton) by placing the insecticidally effective gene part under the control of a light-inducible promoter such as the promoter of the ribulose-1,5-bisphosphate carboxylase small subunit gene of the plant itself or of another plant such as pea as disclosed in U.S. Pat. No. 5,254,799. Another alternative is to use a promoter whose expression is inducible, preferably by wounding such as insect feeding, e.g., the MPI promoter described by Cordera et al. (1994), or the Agrobacterium TR2' or mannopine synthase promoter (Velten et al., 1984) or a promoter inducible by chemical factors.

[0072] The insecticidally effective cry gene part is preferably inserted in the plant genome so that the inserted gene part is upstream (i.e., 5') of suitable 3' end transcription regulation signals (i.e., transcript formation and polyadenylation signals). This is preferably accomplished by inserting the cry1 chimeric gene in the plant cell genome. Preferred polyadenylation and transcript formation signals include those of the 3' untranslated region of the NADP-malic enzyme gene from Flaveria bidentis (Marshall et al., 1996), nopaline synthase gene (Depicker et al., 1982), the octopine synthase gene (Gielen et al., 1984) and the T-DNA gene 7 (Velten and Schell, 1985), which act as 3'-untranslated DNA sequences in transformed plant cells.

[0073] In one embodiment of this invention, at least one of the genes of the invention, preferably at least 2, are transformed into plants selected from the group consisting of: corn, cotton, watercress, horseradish, wasabi, arugula, cress, radish, canola, soybean, vegetable plants, Cruciferae plant species, Brassicaceae plant species such as cauliflower, cabbage, Chinese cabbage, turnip, mustard, oilseed rape, kale, broccoli, Brussels sprouts, mustard spinach, and the like. Particularly, in one embodiment of this invention the following Brassica species plants are protected from insects by the genes of this invention: B. carinata, B. elongate, B. fruticulosa, B. juncea, B. napus, B. narinosa, B. hirta, B. rosularis, B. nigra, B. oleracea, B. perviridis, B. rapa, B. rupestris, B. septiceps, B. tournefortii, and the like, particularly plants of the species Brassica oleraceae (such as the subspecies botrytis and capitate) or Brassica napus, as well as plants of the following genus: Raphanus (such as R. sativus), Armoracia (such as A. rusticana), Wasabia (such as W. japonica), Eruca (such as E. vesicaria), Nastrurtium (such as N. officinale), and Lepidium (such as L. sativum).

[0074] The invention includes the above listed Brassica species plants transformed with at least one or two genes of the invention, such as the cry1B and cry1C genes of the invention, as well as plants obtained after crossing or breeding with related plants (including plants of a related plant species) that contain the genes of the invention. Such crossing or breeding can be done using traditional breeding techniques known in the art, but may also include known in vitro work such as embryo rescue, protoplast fusion, and the like. The invention hence also relates to Brassicaceae plants such as B. napus, B. rapa, B. juncea or B. carinata, that contain the gene or genes of the invention, such as the cry1B and cry1C genes of the invention, from crossings with a transformed B. oleracea plant or the progeny thereof, or to B. oferacea plants that contain the gene or genes of the invention, such as the cry1B and cry1C genes of the invention, from crossings with a transformed B. napus plant, and to uses of such plants.

[0075] Transformation of plant cells can also be used to produce the proteins of the invention in large amounts in plant cell cultures, e.g., to produce a Cry1 protein that can then be applied onto crops after proper formulation. When reference to a transgenic plant cell is made herein, this refers to a plant cell (or also a plant protoplast) as such in isolation or in tissue culture, or to a plant cell (or protoplast) contained in a plant or in a differentiated organ or tissue, and both possibilities are specifically included herein. Hence, a reference to a plant cell in the description or claims is not meant to refer only to isolated cells in culture, but refers to any plant cell, wherever it may be located or in whatever type of plant tissue or organ it may be present.

[0076] All or part of the cry1 genes of the invention, encoding an anti-lepidopteran protein, can also be used to transform bacteria, such as a B. thuringiensis which has insecticidal activity against Lepidoptera or Coleoptera. Thereby, a transformed Bt strain can be produced which is useful for combatting a wide spectrum of lepidopteran and coleopteran insect pests or for combatting additional lepidopteran insect pests. Transformation of bacteria, such as bacteria of the genus Pseudomonas, Agrobacterium, Bacillus or Escherichia, with the cry1 genes of this invention, incorporated in a suitable cloning vehicle, can be carried out in a conventional manner, preferably using conventional electroporation techniques as described in Mahillon et al. (1989) and in PCT Patent publication WO 90/06999.

[0077] Transformed Bacillus species strains containing the cry gene of this invention can be fermented by conventional methods (Dulmage, 1981; Bernhard and Utz, 1993) to provide high yields of cells. Under appropriate conditions which are well understood (Dulmage, 1981), these strains each sporulate to produce crystal proteins containing the Cry protoxin in high yields.

[0078] An insecticidal, particularly anti-lepidopteran, composition of this invention can be formulated in a conventional manner using the microorganisms transformed with the cry gene, or preferably their respective Cry proteins or the Cry protoxin, toxin or insecticidally effective protoxin portion as an active ingredient, together with suitable carriers, diluents, emulsifiers and/or dispersants (e.g., as described by Bernhard and Utz, 1993). This Insecticide composition can be formulated as a wettable powder, pellets, granules or dust or as a liquid formulation with aqueous or non-aqueous solvents as a foam, gel, suspension, concentrate, etc.

[0079] A method for controlling insects, particularly Lepidoptera, in accordance with this invention can comprise applying (e.g., spraying), to a locus (area) to be protected, an insecticidal amount of the Cry proteins or host cells transformed with the ay gene of this invention. The locus to be protected can include, for example, the habitat of the insect pests or growing vegetation or an area where vegetation is to be grown.

[0080] In one embodiment of this invention, insects against which the cry1 genes or Cry1 proteins of the invention can be used include insects selected from the group consisting of: Plutella xylostella, Spodoptera exigua, Spodoptera littoralis, Spodoptera frugiperda, Trichoplusia ni, Heliothis virescens, Mamestra brassicae, Pieris brassicae, Manduca sexta, Choristoneura fumiferana, Choristoneura occidentalis, Choristoneura rosaceana, Pandemis pyrusana, Platynota stultana, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, Lambina fiscellaria, Chilo suppressalis, Chilo partellus, Scirpophaga incertulas, Argyrotaenia citrana, Artogeia rape, Chrysomela scripta, Ostrinia nubilalis, Pseudoplusia includens, and Thaumetopoea pityocampa. In one embodiment, Plutella xylostella (diamondback moth) is a preferred target insect pest. This is a cosmopolitan species that causes major losses in several Cruciferous plants, particularly Brassicacaea plants. The Cry1C, Cry1B and Cry1D proteins encoded by the genes of this invention are particularly useful to control this insect, e.g., by expression of the genes of the invention in cells of a plant.

[0081] Such insects can be controlled by planting or growing plants comprising any one of the cry1C genes of the invention in a field, or by securing the presence of a Cry1C protein as defined herein in or on plants infested by such insects (e.g., by sowing or planting a Brassica species plant such as a cabbage or cauliflower plant transformed with the cry1C1 or cry1C2 gene of this invention, or spraying a composition containing a Cry1C protein of this invention). The invention also relates to the use of the cry1 genes of this invention, at least the cry1C1 or cry1C2 genes, in plants to protect them against Lepidopteran insect pests, preferably in combination with a cry1B or cry1D gene of this invention.

[0082] In the current invention, also a modified coding sequence encoding a chloroplast transit peptide is provided. Such coding sequence has a codon usage adapted for high expression in plants, particularly Brassicaceae plants such as Brassica oleracea or Brassica napus, especially cabbage, cauliflower or oilseed rape (canola). In one embodiment of the invention, the modified transit peptide comprises the nucleotide sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position 371, particularly the sequence of SEQ ID No. 16. Also plant cells, plants or seeds comprising the modified transit peptide coding sequence of the invention, as well as the use of this transit peptide coding sequence for targeting any protein to the chloroplast, particularly to the chloroplast of vegetable plants, particularly Brassica species plants, are included in this invention.

[0083] These and/or other embodiments of this invention are reflected in the wordings of the claims, that form part of the description of the invention.

[0084] The following Examples illustrate the invention, and are not provided to limit the invention or the protection sought. Unless otherwise stated, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.

[0085] The enclosed sequence listing referred to in the Examples, the Claims and the Description is as follows:

[0086] Sequence Listing:

[0087] SEQ ID No.1: optimized cry1C1 coding sequence comprising an intron at position 672

[0088] SEQ ID No.2: amino acid sequence of the Cry1C1 protein encoded by SEQ ID No. 1

[0089] SEQ ID No.3: optimized cry1C2 coding sequence, comprising an intron at position 489

[0090] SEQ ID No.4: optimized cry1C3 coding sequence, comprising the sequences of SEQ ID No. 1 and SEQ ID No. 16, encoding a fusion protein with a transit peptide

[0091] SEQ ID No.5: Cry1C3 protein encoded by SEQ ID No. 4

[0092] SEQ ID No.6: optimized cry1C4 coding sequence, comprising the sequences of SEQ ID

[0093] No. 3 and SEQ ID No. 16, encoding a fusion protein with a transit peptide

[0094] SEQ ID No.7: Cry1C4 protein encoded by SEQ ID No. 6

[0095] SEQ ID No.8: optimized cry1B1 coding sequence, including a transit peptide coding sequence

[0096] SEQ ID No.9: Cry1B1 protein encoded by the sequence of SEQ ID No. 8

[0097] SEQ ID No.10: optimized cry1B2 coding sequence

[0098] SEQ ID No.11. Cry1B2 protein encoded by the sequence of SEQ ID No. 10

[0099] SEQ ID No.12: optimized cry1D1 coding sequence, including a transit peptide coding sequence

[0100] SEQ ID No.13: Cry1D1 protein encoded by the sequence of SEQ ID No.12

[0101] SEQ ID No.14: optimized cry1D2 coding sequence

[0102] SEQ ID No.15: Cry1D2 protein encoded by the sequence of SEQ ID No. 14

[0103] SEQ ID No.16: coding sequence encoding an optimized chloroplast transit peptide

[0104] SEQ ID No.17: chloroplast transit peptide encoded by the sequence of SEQ ID No. 16

[0105] SEQ ID No.18: duplicated S7 subterranean clover stunt virus promoter sequence (S7S7)

[0106] SEQ ID No.19: duplicated S4 subterranean clover stunt virus promoter sequence (S4S4)

[0107] SEQ ID No. 20: cry1B gene primer P1B227

[0108] SEQ ID No. 21: cry1B gene primer P1B228

[0109] SEQ ID No. 22: cry1C gene primer P1C247

[0110] SEQ ID No. 23: cry1C gene primer P1C252

EXAMPLES

1.Construction of Chimeric Genes and Transformation Vectors

[0111] Several cry1 genes were designed and assembled using a combination of technologies to achieve genes with optimal performance in plant cells.

[0112] The cry1C1 DNA which was designed for optimal expression in plant cells is represented in SEQ ID No. 1. This DNA encodes the insecticidal Cry1C1 protein of the invention (SEQ ID No. 2). For transformation of plants, a first chimeric gene (the cry1C1 chimeric gene) is constructed comprising the following operably-linked elements (5' to 3'): a promoter comprising the duplicated promoter region derived from the subterranean clover stunt virus genome segment 7 (S7S7 promoter, Boevink et al., 1995, SEQ ID No. 18), the leader sequence of the tapetum-specific E1 gene (GE1) of Oryza sativa (Michiels et al., 1992), the cry1C1 DNA comprising the second intron of the light-inducible tissue-specific ST-LS1 gene of Solanum tuberosum (Eckes et al., 1986) at position 672 (SEQ ID No. 1), and the sequence including the 3' untranslated region of the NADP-malic enzyme gene from Flaveria bidentis (3' Me1, Marshall et al., 1996).

[0113] A similar cry1C chimeric gene was made, wherein the ST-LS1 intron 2 is at position 489 of the cry1C DNA (i.e., the cry1C2 DNA), this is the cry1C2 chimeric gene, otherwise constructed exactly like the cry1C1 chimeric gene.

[0114] To secure targeting of the Cry1C protein to the plant cell chloroplast, variants of the cry1C1 and cry1C2 chimeric genes are constructed which comprise a modified sequence encoding an optimized transit peptide (SEQ ID No.16) as described by Lebrun et al. (1996) operably-linked to the cry1C coding region so that a transit peptide fusion protein is expressed in plant cells. These are the cry1C3 and cry1C4 chimeric genes, comprising the cry1C3 and cry1C4 coding sequences, respectively, which each contain the sequence of the modified chloroplast transit peptide of SEQ ID No.16. The cry1C3 DNA sequence is shown in SEQ ID No. 4, it is a fusion of the cry1C1 sequence of SEQ ID No. 1 with the transit peptide coding sequence of SEQ ID No. 16. The cry1C4 DNA sequence is shown in SEQ ID No. 6, it is a fusion of the cry1C2 sequence of SEQ ID No. 3 with the transit peptide coding sequence of SEQ ID No. 16.

[0115] The cry1B1 DNA which was designed for optimal expression in plant cells is represented in SEQ ID No. 8. This DNA encodes the insecticidal Cry1B1 protein of the invention (SEQ ID No. 9). For transformation of plants, a chimeric gene (the cry1B1 chimeric gene) is constructed comprising the following operably-linked elements (5' to 3'): a promoter comprising the duplicated promoter region derived from the subterranean clover stunt virus genome segment 4 (S4S4 promoter, Boevink et al., 1995, SEQ ID No. 19), the leader sequence of the E1 gene (GE1) of Oryza sativa (Michiels et al., 1992), the cry1B1 DNA comprising the sequence of the modified chloroplast transit peptide of SEQ ID No.16, and the sequence including the 3' untranslated region of the NADP-malic enzyme gene from Flaveria bidentis (3' Me1, Marshall et al., 1996).

[0116] A second form of the cry1B chimeric gene was also made, using the cry1B2 DNA (SEQ ID No. 10), wherein no sequence encoding an optimized transit peptide is contained, so that cytoplasmic accumulation of the Cry1B protein occurs in plant cells. This is the Cry1B2 chimeric gene.

[0117] The cry1D1 DNA which was designed for optimal expression in plant cells is represented in SEQ ID No. 12. This DNA encodes the insecticidal Cry1D1 protein of the invention (SEQ ID No. 13). For transformation of plants, a chimeric gene (the cry1D1 chimeric gene) is constructed comprising the following operably-linked elements (5' to 3'): an S4S4 promoter (SEQ ID No. 19), the leader sequence of the E1 gene (GE1) of Oryza sativa (Michiels et al., 1992), the cry1D1 DNA comprising the sequence of the modified chloroplast transit peptide of SEQ ID No.16, and the sequence including the 3' untranslated region of the NADP-malic enzyme gene from Flaveria bidentis (3' Me1, Marshall et al., 1996).

[0118] A second form of the cry1D chimeric gene was also made, using the cry1D2 DNA, wherein no sequence encoding an optimized transit peptide is contained, so that cytoplasmic accumulation of the Cry1D protein occurs in plant cells. This is the Cry1D2 chimeric gene.

[0119] A DNA transformation vector (pT1C4B1) is made comprising between the T-DNA borders the cry1C4 chimeric gene and the cry1B1 chimeric gene in a head-to-tail orientation (3'Me1-cry1C4-GE1 leader-S7S7-S4S4-GE1 leader-cry1B1-3'Me1), as well as a transfer vector (pT1C2B2) comprising between the T-DNA borders the cry1C2 chimeric gene and the cry1B2 chimeric gene in a head-to-tail orientation (3'Me1-cry1C2-GE1 leader-S7S7-S4S4-GE1 leader-cry1B2-3'Me1). In such manner, with both T-DNA vectors, the cry1C and cry1B genes of the invention will be co-transferred to the plant cell and will be located at one locus after successful transformation.

[0120] Similar T-DNA vectors are constructed which contain the above cry1C chimeric genes but which contain as second chimeric gene the cry1D1 or cry1D2 chimeric genes instead of the above cry1B chimeric genes. Also a triple cry gene transformation vector is constructed, comprising both the cry1C, cry1D and cry1B genes (all either with or without modified transit peptide).

[0121] The transformation vectors containing the genes of the invention were derived from pGSC1700 (Cornelissen and Vandewiele, 1989). The vector backbone contains the following genetic elements:

[0122] a) the plasmid core comprising the origin of replication from the plasmid pBR322 (Bolivar et al., 1977) for replication in Escherichia coli and a restriction fragment comprising the origin of replication from the Pseudomonas plasmid pVS1 (itoh et al., 1984) for replication in Agrobacterium tumefaciens.

[0123] b) a selectable marker gene conferring resistance to streptomycin and spectinomycin (aadA) for propagation and selection of the plasmid in Escherichia coli and Agrobacterium tumefaciens.

[0124] c) a DNA region consisting of a fragment of the neomycin phosphotransferase coding sequence of the nptl gene from transposon Tn903 (Oka et al., 1981).

[0125] The T-DNA region of each transformation vector also contains a chimeric bar gene that serves as selectable marker gene. Expression of the bar gene enables the production of an enzyme, phosphinothricin-acetyl transferase, that metabolizes the herbicide glufosinate-ammonium, thus rendering it non-herbicidal in the plant. The chimeric bar gene comprises the 35S3 promoter region from the Cauliflower Mosaic Virus 35S transcript (Odell et al.,1985), the bar coding sequence of the phosphinothricin acetyltransferase gene of Streptomyces hygroscopicus as described by Thompson et al. (1987), and a 3' transcript termination and polyadenylation sequence from the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982).

[0126] Similar transformation vectors as those described are also constructed, wherein the cry1C1 or cry1C3 chimeric genes are used (similar as the above cry1C genes but having the ST-LS1 intron at a position 489). Also these vectors contain the cry1B1 or cry1B2 chimeric genes, or the cry1D1 or cry1D2 chimeric genes described above.

[0127] All constructed plasmids are confirmed to be accurate by restriction enzyme digest analysis and by DNA sequencing, before they are used for plant transformation.

2.Plant Transformation and Regeneration

[0128] The above transformation vectors pT1C4B1 and pT1C2B2 containing the cry1C and cry1B genes of the invention are transferred into Agrobacterium tumefaciens strains for transformation in plants using routine methods.

[0129] Cauliflower and cabbage plants are transformed using Agrobacterium transformation. Seeds of Brassica oleracea var. capitata (cabbage) or Brassica oleracea var. botrytis (cauliflower) are sterilized by dipping in 70% ethanol followed by submersion in 6% bleach. The seeds are then rinsed with sterile water and transferred to small Petri-plates containing MS based medium. The Petri-plates are placed in glass containers and incubated for 5-8 days at 24.degree. C. Hypocotyl explants of 0.5-0.7 cm are cut and placed in liquid medium with appropriate hormones. Agrobacterium tumefaciens carrying the genes of interest are added to the medium to make a final concentration of 1.times.10.sup.7 bacteria/ml. After the co-cultivation period, the explants are washed in liquid medium with appropriate antibiotics and hormones and blotted dry on filter paper.

[0130] The explants are cultured for one week on callus induction medium with 5 mg/l silver nitrate and 250 mg/l of both Triacillin and Carbenicillin and 10 mg/l phosphinothricin for selection of transformation events.

[0131] Every two weeks explants are transferred to fresh medium. Every week explants are checked for callus formation. Calli are excised from the explants and transferred to shoot induction medium. Shoots are transferred to plastic containers with rooting medium. Shoots are kept on this medium until they are normalized or rooted. If they are 3-10 cm in size and have a nicely developed root system, they are transferred to the greenhouse.

[0132] Oilseed rape plants are also transformed with the cry1C and cry1B genes using Agrobacterium tumefaciens. Hypocotyl explants of Brassica napus are used in routine transformation and regeneration methods, e.g., the method described by De Block et al. (1989).

3. Analysis of Transformants

[0133] Once the transformed plants are regenerated, PCR and Southern analysis are used to confirm integration of the transgenes. Immunological analyses such as Cry1C- and Cry1B-specific ELISA assays or Western blots are used to select those transformed plants showing optimal expression levels of the Cry1C and Cry1B proteins.

[0134] RT-PCR experiments on RNA collected from cauliflower plants shown to be transformed with the cry1C genes of SEQ ID No. 1 or 3, comprising a plant intron at a different position, confirmed that splicing occurs correctly and that a functional Cry1C protein is produced in these plants. This is also confirmed by Northern blot analysis of these plants.

[0135] Also, insect assays using Plutella xylostella larvae under standard insect bio-assay conditions using proper controls with selected transformed cabbage, cauliflower and oilseed rape plants containing the Cry1C and Cry1B genes confirm the high insecticidal activity and the high dose of these protein expressed, in those transformed plants selected for optimal expression. Also, Plutella xylostella insects that have been selected for resistance to the Cry1C or Cry1B protein, are still effectively killed by the plants of the invention.

[0136] Progeny plants and seeds are also obtained from the transformed, selected plants of the invention, and the genes of the invention are shown to segregate in such progeny in the expected Mendelian fashion. Selection of the transgenic plants in the greenhouse and in the field at multiple locations will result in the identification of plant lines which have optimal stability and expression of the cry1 chimeric genes combined with optimal agronomical performance. Crossing of the selected best performing transgenic plants with several different commercial lines, and repeated backcrossing therewith, result in the presence of the (linked) cry1B and cry1C genes of the invention in different cabbage, cauliflower or oilseed rape genetic backgrounds, optimally adapted to different areas or climatic conditions.

CITED REFERENCES

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[0211] All cited references are hereby incorporated by reference into the description. The citation of any of these references is not to be construed as an acknowledgement of the accuracy of every statement contained in such reference, nor as an acknowledgement that such reference is relevant prior art or part of the common general knowledge in any territory.

Sequence CWU 1

1

2312076DNAArtificial SequenceOptimized cry1C1 coding sequence 1atg gct gag gag aac aac cag aac cag tgt atc cct tac aac tgt ctt 48Met Ala Glu Glu Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu1 5 10 15tcg aac cct gag gag gtt ctt ctt gat gga gag aga atc tct act gga 96Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly20 25 30aac tct tct atc gat att tct ctt tct ctt gtt cag ttc ctt gtt tct 144Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser35 40 45aac ttc gtt cct gga gga gga ttc ctt gtt gga ctt atc gat ttc gtt 192Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val50 55 60tgg gga atc gtt gga cct tct cag tgg gat gct ttc ctt gtt cag atc 240Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile65 70 75 80gag cag ctt atc aac gag aga atc gct gag ttc gct aga aac gct gct 288Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala85 90 95atc gct aac ctt gag gga ctt gga aac aac ttc aac atc tac gtt gag 336Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu100 105 110gct ttc aag gag tgg gag gag gat cct aac aac cct gag act aga act 384Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr115 120 125aga gtt atc gat aga ttc aga atc ctt gat gga ctt ctt gag aga gat 432Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp130 135 140att cct tct ttc aga atc tct gga ttc gag gtt cct ctt ctt tct gtt 480Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val145 150 155 160tac gct cag gct gct aac ctt cat ctt gct atc ctt aga gat tct gtt 528Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val165 170 175atc ttc gga gag aga tgg gga ctt act act atc aac gtt aac gag aac 576Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn180 185 190tac aac aga ctt atc aga cat atc gat gag tac gct gat cat tgt gct 624Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala195 200 205aac act tac aac aga gga ctt aac aac ctt cct aag tct act tac cag 672Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln210 215 220gtaagtttct gcttctacct ttgatatata tataataatt atcattaatt agtagtaata 732taatatttca aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg 792tagtttataa gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt 852gatgtgcag gac tgg atc act tac aac aga ctt aga aga gat ctt act ctt 903Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu225 230 235act gtt ctt gat att gct gct ttc ttc cct aac tac gat aac aga aga 951Thr Val Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg240 245 250tac cct atc cag cct gtt gga cag ctt act aga gag gtt tac act gat 999Tyr Pro Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp255 260 265 270cct ctt atc aac ttc aac cct cag ctt cag tct gtt gct cag ctt cct 1047Pro Leu Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro275 280 285act ttc aac gtt atg gag tct tct gct atc aga aac cct cat ctt ttc 1095Thr Phe Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe290 295 300gat att ctt aac aac ctt act atc ttc act gac tgg ttc tct gtt gga 1143Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly305 310 315aga aac ttc tac tgg gga gga cat aga gtt atc tct tct ctt atc gga 1191Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly320 325 330gga gga aac atc act tct cct atc tac gga aga gag gct aac cag gag 1239Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu335 340 345 350cct cct aga tct ttc act ttc aac gga cct gtt ttc aga act ctt tct 1287Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser355 360 365aac cct act ctt aga ctt ctt cag cag cct tgg cct gct cct cct ttc 1335Asn Pro Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe370 375 380aac ctt aga gga gtt gag gga gtt gag ttc tct act cct act aac tct 1383Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser385 390 395ttc act tac aga gga aga gga act gtt gat tct ctt act gag ctt cct 1431Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro400 405 410cct gag gat aac tct gtt cct cct aga gag gga tac tct cat aga ctt 1479Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu415 420 425 430tgt cat gct act ttc gtt cag aga tct gga act cct ttc ctt act act 1527Cys His Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr435 440 445gga gtt gtt ttc tct tgg act cat aga tct gct act ctt act aac act 1575Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr450 455 460atc gat cct gag agg atc aac cag atc cct ctt gtt aag gga ttc aga 1623Ile Asp Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg465 470 475gtt tgg gga gga act tct gtt atc act gga cct gga ttc act gga gga 1671Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly480 485 490gat att ctt aga aga aac act ttc gga gat ttc gtt tct ctt cag gtt 1719Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val495 500 505 510aac atc aac tct cct atc act cag aga tac aga ctt aga ttc aga tac 1767Asn Ile Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr515 520 525gct tct tct aga gat gct aga gtt atc gtt ctt act gga gct gct tct 1815Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser530 535 540act gga gtt gga gga cag gtt tct gtt aac atg cct ctt cag aag act 1863Thr Gly Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr545 550 555atg gag atc gga gag aac ctt act tct aga act ttc aga tac act gat 1911Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp560 565 570ttc tct aac cct ttc tct ttc aga gct aac cct gat att atc gga atc 1959Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile575 580 585 590tct gag cag cct ctt ttc gga gct gga tct atc tct tct gga gag ctt 2007Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu595 600 605tac atc gat aaa atc gag atc atc ctt gct gat gct act ttc gag gct 2055Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala610 615 620gag tct gat tta gag aga tga 2076Glu Ser Asp Leu Glu Arg6252628PRTArtificial SequenceCryC1 protein 2Met Ala Glu Glu Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu1 5 10 15Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly20 25 30Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser35 40 45Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val50 55 60Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile65 70 75 80Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala85 90 95Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu100 105 110Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr115 120 125Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp130 135 140Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val145 150 155 160Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val165 170 175Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn180 185 190Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala195 200 205Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln210 215 220Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val225 230 235 240Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro245 250 255Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu260 265 270Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe275 280 285Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile290 295 300Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn305 310 315 320Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly325 330 335Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro340 345 350Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro355 360 365Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu370 375 380Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr385 390 395 400Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu405 410 415Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His420 425 430Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val435 440 445Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp450 455 460Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp465 470 475 480Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile485 490 495Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile500 505 510Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser515 520 525Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly530 535 540Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu545 550 555 560Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser565 570 575Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu580 585 590Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile595 600 605Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser610 615 620Asp Leu Glu Arg62532076DNAArtificial SequenceOptimized cry1C2 coding sequence 3atg gct gag gag aac aac cag aac cag tgt atc cct tac aac tgt ctt 48Met Ala Glu Glu Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu1 5 10 15tcg aac cct gag gag gtt ctt ctt gat gga gag aga atc tct act gga 96Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly20 25 30aac tct tct atc gat att tct ctt tct ctt gtt cag ttc ctt gtt tct 144Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser35 40 45aac ttc gtt cct gga gga gga ttc ctt gtt gga ctt atc gat ttc gtt 192Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val50 55 60tgg gga atc gtt gga cct tct cag tgg gat gct ttc ctt gtt cag atc 240Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile65 70 75 80gag cag ctt atc aac gag aga atc gct gag ttc gct aga aac gct gct 288Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala85 90 95atc gct aac ctt gag gga ctt gga aac aac ttc aac atc tac gtt gag 336Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu100 105 110gct ttc aag gag tgg gag gag gat cct aac aac cct gag act aga act 384Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr115 120 125aga gtt atc gat aga ttc aga atc ctt gat gga ctt ctt gag aga gat 432Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp130 135 140att cct tct ttc aga atc tct gga ttc gaa gtt cct ctt ctt tct gtt 480Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val145 150 155 160tac gct cag gtaagtttct gcttctacct ttgatatata tataataatt 529Tyr Ala Glnatcattaatt agtagtaata taatatttca aatatttttt tcaaaataaa agaatgtagt 589atatagcaat tgcttttctg tagtttataa gtgtgtatat tttaatttat aacttttcta 649atatatgacc aaaacatggt gatgtgcag gct gct aac ctt cat ctt gct atc 702Ala Ala Asn Leu His Leu Ala Ile165 170ctt aga gat tct gtt atc ttc gga gag aga tgg gga ctt act act atc 750Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile175 180 185aac gtt aac gag aac tac aac aga ctt atc aga cat atc gat gag tac 798Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr190 195 200gct gat cat tgt gct aac act tac aac aga gga ctt aac aac ctt cct 846Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro205 210 215aag tct act tac cag gac tgg atc act tac aac aga ctt aga aga gat 894Lys Ser Thr Tyr Gln Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp220 225 230 235ctt act ctt act gtt ctt gat att gct gct ttc ttc cct aac tac gat 942Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp240 245 250aac aga aga tac cct atc cag cct gtt gga cag ctt act aga gag gtt 990Asn Arg Arg Tyr Pro Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val255 260 265tac act gat cct ctt atc aac ttc aac cct cag ctt cag tct gtt gct 1038Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala270 275 280cag ctt cct act ttc aac gtt atg gag tct tct gct atc aga aac cct 1086Gln Leu Pro Thr Phe Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro285 290 295cat ctt ttc gat att ctt aac aac ctt act atc ttc act gac tgg ttc 1134His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe300 305 310 315tct gtt gga aga aac ttc tac tgg gga gga cat aga gtt atc tct tct 1182Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser320 325 330ctt atc gga gga gga aac atc act tct cct atc tac gga aga gag gct 1230Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala335 340 345aac cag gag cct cct aga tct ttc act ttc aac gga cct gtt ttc aga 1278Asn Gln Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg350 355 360act ctt tct aac cct act ctt aga ctt ctt cag cag cct tgg cct gct 1326Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala365 370 375cct cct ttc aac ctt aga gga gtt gag gga gtt gag ttc tct act cct 1374Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro380 385 390 395act aac tct ttc act tac aga gga aga gga act gtt gat tct ctt act 1422Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr400 405 410gag ctt cct cct gag gat aac tct gtt cct cct aga gag gga tac tct 1470Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser415 420 425cat aga ctt tgt cat gct act ttc gtt cag aga tct gga act cct ttc 1518His Arg Leu Cys His Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe430 435 440ctt act act gga gtt gtt ttc tct tgg act cat aga tct gct act ctt 1566Leu Thr Thr Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu445 450 455act aac act atc gat cct gag agg atc aac cag atc cct ctt gtt aag 1614Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys460 465 470 475gga ttc aga gtt tgg gga gga act tct gtt atc act gga cct gga ttc 1662Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe480 485 490act gga gga gat att ctt aga aga aac act ttc gga gat ttc gtt tct 1710Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser495 500 505ctt cag gtt aac atc aac tct cct atc act cag aga tac aga ctt aga 1758Leu Gln Val Asn Ile Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg510 515 520ttc aga tac gct tct tct aga gat gct aga gtt atc gtt ctt act gga 1806Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly525 530 535gct gct tct act gga gtt gga gga cag gtt tct

gtt aac atg cct ctt 1854Ala Ala Ser Thr Gly Val Gly Gly Gln Val Ser Val Asn Met Pro Leu540 545 550 555cag aag act atg gag atc gga gag aac ctt act tct aga act ttc aga 1902Gln Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg560 565 570tac act gat ttc tct aac cct ttc tct ttc aga gct aac cct gat att 1950Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile575 580 585atc gga atc tct gag cag cct ctt ttc gga gct gga tct atc tct tct 1998Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser590 595 600gga gag ctt tac atc gat aaa atc gag atc atc ctt gct gat gct act 2046Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr605 610 615ttc gag gct gag tct gat tta gag aga tga 2076Phe Glu Ala Glu Ser Asp Leu Glu Arg620 62542442DNAArtificial SequenceOptimized cry1C3 coding sequence 4atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro1 5 10 15gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala20 25 30gct ttc cct act act aag aag gct aac gat ttc tct act ctt cct tct 144Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser35 40 45aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac 192Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn50 55 60aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct 240Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro65 70 75 80act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga 288Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly85 90 95ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct 336Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser100 105 110ctt gga aac gtt tct aac gga gga aga atc aga tgt gag gag aac aac 384Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn115 120 125cag aac cag tgt atc cct tac aac tgt ctt tcg aac cct gag gag gtt 432Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val130 135 140ctt ctt gat gga gag aga atc tct act gga aac tct tct atc gat att 480Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile145 150 155 160tct ctt tct ctt gtt cag ttc ctt gtt tct aac ttc gtt cct gga gga 528Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly165 170 175gga ttc ctt gtt gga ctt atc gat ttc gtt tgg gga atc gtt gga cct 576Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro180 185 190tct cag tgg gat gct ttc ctt gtt cag atc gag cag ctt atc aac gag 624Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Glu195 200 205aga atc gct gag ttc gct aga aac gct gct atc gct aac ctt gag gga 672Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly210 215 220ctt gga aac aac ttc aac atc tac gtt gag gct ttc aag gag tgg gag 720Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu225 230 235 240gag gat cct aac aac cct gag act aga act aga gtt atc gat aga ttc 768Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe245 250 255aga atc ctt gat gga ctt ctt gag aga gat att cct tct ttc aga atc 816Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile260 265 270tct gga ttc gag gtt cct ctt ctt tct gtt tac gct cag gct gct aac 864Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Ala Asn275 280 285ctt cat ctt gct atc ctt aga gat tct gtt atc ttc gga gag aga tgg 912Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp290 295 300gga ctt act act atc aac gtt aac gag aac tac aac aga ctt atc aga 960Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg305 310 315 320cat atc gat gag tac gct gat cat tgt gct aac act tac aac aga gga 1008His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly325 330 335ctt aac aac ctt cct aag tct act tac cag gtaagtttct gcttctacct 1058Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln340 345ttgatatata tataataatt atcattaatt agtagtaata taatatttca aatatttttt 1118tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat 1178tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcag gac tgg atc 1236Asp Trp Ileact tac aac aga ctt aga aga gat ctt act ctt act gtt ctt gat att 1284Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile350 355 360 365gct gct ttc ttc cct aac tac gat aac aga aga tac cct atc cag cct 1332Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro370 375 380gtt gga cag ctt act aga gag gtt tac act gat cct ctt atc aac ttc 1380Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe385 390 395aac cct cag ctt cag tct gtt gct cag ctt cct act ttc aac gtt atg 1428Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met400 405 410gag tct tct gct atc aga aac cct cat ctt ttc gat att ctt aac aac 1476Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn415 420 425ctt act atc ttc act gac tgg ttc tct gtt gga aga aac ttc tac tgg 1524Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp430 435 440 445gga gga cat aga gtt atc tct tct ctt atc gga gga gga aac atc act 1572Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr450 455 460tct cct atc tac gga aga gag gct aac cag gag cct cct aga tct ttc 1620Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe465 470 475act ttc aac gga cct gtt ttc aga act ctt tct aac cct act ctt aga 1668Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg480 485 490ctt ctt cag cag cct tgg cct gct cct cct ttc aac ctt aga gga gtt 1716Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val495 500 505gag gga gtt gag ttc tct act cct act aac tct ttc act tac aga gga 1764Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly510 515 520 525aga gga act gtt gat tct ctt act gag ctt cct cct gag gat aac tct 1812Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser530 535 540gtt cct cct aga gag gga tac tct cat aga ctt tgt cat gct act ttc 1860Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe545 550 555gtt cag aga tct gga act cct ttc ctt act act gga gtt gtt ttc tct 1908Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser560 565 570tgg act cat aga tct gct act ctt act aac act atc gat cct gag agg 1956Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg575 580 585atc aac cag atc cct ctt gtt aag gga ttc aga gtt tgg gga gga act 2004Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr590 595 600 605tct gtt atc act gga cct gga ttc act gga gga gat att ctt aga aga 2052Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg610 615 620aac act ttc gga gat ttc gtt tct ctt cag gtt aac atc aac tct cct 2100Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro625 630 635atc act cag aga tac aga ctt aga ttc aga tac gct tct tct aga gat 2148Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp640 645 650gct aga gtt atc gtt ctt act gga gct gct tct act gga gtt gga gga 2196Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly655 660 665cag gtt tct gtt aac atg cct ctt cag aag act atg gag atc gga gag 2244Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu670 675 680 685aac ctt act tct aga act ttc aga tac act gat ttc tct aac cct ttc 2292Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe690 695 700tct ttc aga gct aac cct gat att atc gga atc tct gag cag cct ctt 2340Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu705 710 715ttc gga gct gga tct atc tct tct gga gag ctt tac atc gat aaa atc 2388Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile720 725 730gag atc atc ctt gct gat gct act ttc gag gct gag tct gat tta gag 2436Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu735 740 745aga tga 2442Arg7505750PRTArtificial SequenceCry1C3 protein 5Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro1 5 10 15Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala20 25 30Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser35 40 45Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn50 55 60Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro65 70 75 80Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly85 90 95Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser100 105 110Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn115 120 125Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val130 135 140Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile145 150 155 160Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly165 170 175Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro180 185 190Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Glu195 200 205Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly210 215 220Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu225 230 235 240Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe245 250 255Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile260 265 270Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Ala Asn275 280 285Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp290 295 300Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg305 310 315 320His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly325 330 335Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp Ile Thr Tyr Asn340 345 350Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe355 360 365Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro Val Gly Gln370 375 380Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gln385 390 395 400Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met Glu Ser Ser405 410 415Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile420 425 430Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His435 440 445Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile450 455 460Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe Thr Phe Asn465 470 475 480Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gln485 490 495Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val500 505 510Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr515 520 525Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro530 535 540Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gln Arg545 550 555 560Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His565 570 575Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gln580 585 590Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile595 600 605Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe610 615 620Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro Ile Thr Gln625 630 635 640Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val645 650 655Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gln Val Ser660 665 670Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn Leu Thr675 680 685Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg690 695 700Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala705 710 715 720Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile725 730 735Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg740 745 75062442DNAArtificial SequenceOptimized cry1C4 coding sequence 6atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro1 5 10 15gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala20 25 30gct ttc cct act act aag aag gct aac gat ttc tct act ctt cct tct 144Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser35 40 45aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac 192Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn50 55 60aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct 240Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro65 70 75 80act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga 288Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly85 90 95ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct 336Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser100 105 110ctt gga aac gtt tct aac gga gga aga atc aga tgt gag gag aac aac 384Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn115 120 125cag aac cag tgt atc cct tac aac tgt ctt tcg aac cct gag gag gtt 432Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val130 135 140ctt ctt gat gga gag aga atc tct act gga aac tct tct atc gat att 480Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile145 150 155 160tct ctt tct ctt gtt cag ttc ctt gtt tct aac ttc gtt cct gga gga 528Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly165 170 175gga ttc ctt gtt gga ctt atc gat ttc gtt tgg gga atc gtt gga cct 576Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro180 185 190tct cag tgg gat gct ttc ctt gtt cag atc gag cag ctt atc aac gag 624Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Glu195 200 205aga atc gct gag ttc gct aga aac gct gct atc gct aac ctt gag gga 672Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly210 215 220ctt gga aac aac ttc aac atc tac gtt gag gct ttc aag gag tgg gag 720Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu225 230 235 240gag gat cct aac aac cct gag act aga act aga gtt atc gat aga ttc 768Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe245 250 255aga atc ctt gat gga ctt ctt gag aga gat att cct tct ttc aga atc 816Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile260 265 270tct

gga ttc gaa gtt cct ctt ctt tct gtt tac gct cag gtaagtttct 865Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gln275 280 285gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca 925aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa 985gtgtgtatat tttaatttat aacttttcta atatatgacc aaaacatggt gatgtgcag 1044gct gct aac ctt cat ctt gct atc ctt aga gat tct gtt atc ttc gga 1092Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly290 295 300gag aga tgg gga ctt act act atc aac gtt aac gag aac tac aac aga 1140Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg305 310 315ctt atc aga cat atc gat gag tac gct gat cat tgt gct aac act tac 1188Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr320 325 330aac aga gga ctt aac aac ctt cct aag tct act tac cag gac tgg atc 1236Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp Ile335 340 345act tac aac aga ctt aga aga gat ctt act ctt act gtt ctt gat att 1284Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile350 355 360 365gct gct ttc ttc cct aac tac gat aac aga aga tac cct atc cag cct 1332Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro370 375 380gtt gga cag ctt act aga gag gtt tac act gat cct ctt atc aac ttc 1380Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe385 390 395aac cct cag ctt cag tct gtt gct cag ctt cct act ttc aac gtt atg 1428Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met400 405 410gag tct tct gct atc aga aac cct cat ctt ttc gat att ctt aac aac 1476Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn415 420 425ctt act atc ttc act gac tgg ttc tct gtt gga aga aac ttc tac tgg 1524Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp430 435 440 445gga gga cat aga gtt atc tct tct ctt atc gga gga gga aac atc act 1572Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr450 455 460tct cct atc tac gga aga gag gct aac cag gag cct cct aga tct ttc 1620Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe465 470 475act ttc aac gga cct gtt ttc aga act ctt tct aac cct act ctt aga 1668Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg480 485 490ctt ctt cag cag cct tgg cct gct cct cct ttc aac ctt aga gga gtt 1716Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val495 500 505gag gga gtt gag ttc tct act cct act aac tct ttc act tac aga gga 1764Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly510 515 520 525aga gga act gtt gat tct ctt act gag ctt cct cct gag gat aac tct 1812Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser530 535 540gtt cct cct aga gag gga tac tct cat aga ctt tgt cat gct act ttc 1860Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe545 550 555gtt cag aga tct gga act cct ttc ctt act act gga gtt gtt ttc tct 1908Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser560 565 570tgg act cat aga tct gct act ctt act aac act atc gat cct gag agg 1956Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg575 580 585atc aac cag atc cct ctt gtt aag gga ttc aga gtt tgg gga gga act 2004Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr590 595 600 605tct gtt atc act gga cct gga ttc act gga gga gat att ctt aga aga 2052Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg610 615 620aac act ttc gga gat ttc gtt tct ctt cag gtt aac atc aac tct cct 2100Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro625 630 635atc act cag aga tac aga ctt aga ttc aga tac gct tct tct aga gat 2148Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp640 645 650gct aga gtt atc gtt ctt act gga gct gct tct act gga gtt gga gga 2196Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly655 660 665cag gtt tct gtt aac atg cct ctt cag aag act atg gag atc gga gag 2244Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu670 675 680 685aac ctt act tct aga act ttc aga tac act gat ttc tct aac cct ttc 2292Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe690 695 700tct ttc aga gct aac cct gat att atc gga atc tct gag cag cct ctt 2340Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu705 710 715ttc gga gct gga tct atc tct tct gga gag ctt tac atc gat aaa atc 2388Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile720 725 730gag atc atc ctt gct gat gct act ttc gag gct gag tct gat tta gag 2436Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu735 740 745aga tga 2442Arg7507750PRTArtificial SequenceCry1C4 protein 7Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro1 5 10 15Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala20 25 30Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser35 40 45Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn50 55 60Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro65 70 75 80Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly85 90 95Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser100 105 110Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn115 120 125Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val130 135 140Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile145 150 155 160Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly165 170 175Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro180 185 190Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln Leu Ile Asn Glu195 200 205Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly210 215 220Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu225 230 235 240Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe245 250 255Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile260 265 270Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Ala Asn275 280 285Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp290 295 300Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg305 310 315 320His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly325 330 335Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp Trp Ile Thr Tyr Asn340 345 350Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe355 360 365Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gln Pro Val Gly Gln370 375 380Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gln385 390 395 400Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn Val Met Glu Ser Ser405 410 415Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile420 425 430Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His435 440 445Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile450 455 460Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg Ser Phe Thr Phe Asn465 470 475 480Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gln485 490 495Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val500 505 510Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr515 520 525Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro530 535 540Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gln Arg545 550 555 560Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His565 570 575Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gln580 585 590Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile595 600 605Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe610 615 620Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn Ser Pro Ile Thr Gln625 630 635 640Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val645 650 655Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gln Val Ser660 665 670Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn Leu Thr675 680 685Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg690 695 700Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala705 710 715 720Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile725 730 735Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg740 745 75082313DNAArtificial SequenceOptimized cry1B1 coding sequence 8atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro1 5 10 15gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala20 25 30gct ttc cct act act aag aag gct aac gat ttc tct act ctt cct tct 144Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser35 40 45aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac 192Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn50 55 60aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct 240Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro65 70 75 80act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga 288Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly85 90 95ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct 336Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser100 105 110ctt gga aac gtt tct aac gga gga aga atc aga tgt act tcg aac aga 384Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Thr Ser Asn Arg115 120 125aag aac gag aac gag atc atc aac gct gtt tct aac cat tct gct cag 432Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser Asn His Ser Ala Gln130 135 140atg gat ctt ctt cct gat gct aga atc gag gat tct ctt tgt atc gct 480Met Asp Leu Leu Pro Asp Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala145 150 155 160gag gga aac aac atc gat cct ttc gtt tct gct tct act gtt cag act 528Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Ala Ser Thr Val Gln Thr165 170 175ggt atc aac atc gct gga aga att ctt gga gtt ctt gga gtt cct ttc 576Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Val Leu Gly Val Pro Phe180 185 190gct gga cag ctt gct tct ttc tac tct ttc ctt gtt gga gag ctt tgg 624Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp195 200 205cct aga gga aga gat cag tgg gag atc ttc ctt gag cat gtt gag cag 672Pro Arg Gly Arg Asp Gln Trp Glu Ile Phe Leu Glu His Val Glu Gln210 215 220ctt atc aac cag cag atc act gag aac gct aga aac act gct ctt gct 720Leu Ile Asn Gln Gln Ile Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala225 230 235 240aga ctt cag gga ctt gga gat tct ttc aga gct tac cag cag tct ctt 768Arg Leu Gln Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gln Gln Ser Leu245 250 255gag gac tgg ctt gag aac aga gat gat gct aga act aga tct gtt ctt 816Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg Thr Arg Ser Val Leu260 265 270cat act cag tac atc gct ctt gag ctt gat ttc ctt aac gct atg cct 864His Thr Gln Tyr Ile Ala Leu Glu Leu Asp Phe Leu Asn Ala Met Pro275 280 285ctt ttc gct atc aga aac cag gag gtt cct ctt ctt atg gtt tac gct 912Leu Phe Ala Ile Arg Asn Gln Glu Val Pro Leu Leu Met Val Tyr Ala290 295 300cag gct gct aac ctt cat ctt ctt ctt ctt aga gat gct tct ctt ttc 960Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe305 310 315 320gga tct gag ttc gga ctt act tct cag gag atc cag aga tat tac gag 1008Gly Ser Glu Phe Gly Leu Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu325 330 335aga cag gtt gag aga act aga gat tac tct gat tac tgt gtt gag tgg 1056Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser Asp Tyr Cys Val Glu Trp340 345 350tac aac act gga ctt aac tct ctt aga gga act aac gct gct tct tgg 1104Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr Asn Ala Ala Ser Trp355 360 365gtt aga tac aac cag ttc aga aga gat ctt act ctt gga gtt ctt gat 1152Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr Leu Gly Val Leu Asp370 375 380ctt gtt gct ctt ttc cct tct tac gac act aga act tac cct atc aac 1200Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn385 390 395 400act tct gct cag ctt act aga gag gtt tac act gat gct atc gga gct 1248Thr Ser Ala Gln Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Ala405 410 415act gga gtt aac atg gct tct atg aac tgg tac aac aac aac gct cct 1296Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro420 425 430tct ttc tct gct atc gag gct gct gct atc aga tct cct cat ctt ctt 1344Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile Arg Ser Pro His Leu Leu435 440 445gat ttc ctt gag cag ctt act atc ttc tct gct tct tct aga tgg tct 1392Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser Ala Ser Ser Arg Trp Ser450 455 460aac act aga cac atg act tac tgg aga gga cat acc atc cag tct aga 1440Asn Thr Arg His Met Thr Tyr Trp Arg Gly His Thr Ile Gln Ser Arg465 470 475 480cct atc gga gga gga ctt aac act tct act cat gga gct act aac act 1488Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr485 490 495tct atc aac cct gtt act ctt aga ttc gct tct aga gat gtt tac aga 1536Ser Ile Asn Pro Val Thr Leu Arg Phe Ala Ser Arg Asp Val Tyr Arg500 505 510act gag tct tac gct gga gtt ctt ctt tgg gga atc tac ctt gag cct 1584Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly Ile Tyr Leu Glu Pro515 520 525atc cac gga gtt cct act gtt aga ttc aac ttc act aac cct cag aac 1632Ile His Gly Val Pro Thr Val Arg Phe Asn Phe Thr Asn Pro Gln Asn530 535 540atc tct gat aga gga act gct aac tac tct cag cct tac gag tct cct 1680Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gln Pro Tyr Glu Ser Pro545 550 555 560gga ctt cag ctt aag gat tct gag act gag ctt cct cct gag act act 1728Gly Leu Gln Leu Lys Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr565 570 575gag aga cct aac tac gag tct tac tct cat aga ctt tct cat atc gga 1776Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly580 585 590atc atc ctt cag tct aga gtt aac gtt cct gtt tac tct tgg act cat 1824Ile Ile Leu Gln Ser Arg Val Asn Val Pro Val Tyr Ser Trp Thr His595 600 605aga tct gct gat aga act aac act atc gga cct aac aga atc act cag 1872Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pro Asn Arg Ile Thr Gln610 615 620atc cct atg gtt aag gct tct gag ctt cct cag gga act act gtt gtt 1920Ile Pro Met Val Lys Ala Ser Glu Leu Pro Gln Gly Thr Thr Val Val625 630 635

640aga gga cct gga ttc act gga gga gat atc ctt aga aga act aac act 1968Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr645 650 655gga gga ttc gga cct atc aga gtt act gtt aac gga cct ctt act cag 2016Gly Gly Phe Gly Pro Ile Arg Val Thr Val Asn Gly Pro Leu Thr Gln660 665 670aga tac aga atc gga ttc aga tac gct tct act gtt gat ttc gat ttc 2064Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe675 680 685ttc gtt tct aga gga gga act act gtt aac aac ttc aga ttc ctt aga 2112Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn Phe Arg Phe Leu Arg690 695 700act atg aac tct gga gat gag ctt aag tac gga aac ttc gtt aga aga 2160Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly Asn Phe Val Arg Arg705 710 715 720gct ttc act act cct ttc act ttc act cag atc cag gat atc atc aga 2208Ala Phe Thr Thr Pro Phe Thr Phe Thr Gln Ile Gln Asp Ile Ile Arg725 730 735act tct atc cag gga ctt tct gga aac gga gag gtt tac atc gat aaa 2256Thr Ser Ile Gln Gly Leu Ser Gly Asn Gly Glu Val Tyr Ile Asp Lys740 745 750atc gag atc atc cct gtt act gct act ttc gag gct gag tac gat tta 2304Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu755 760 765gag aga tga 2313Glu Arg7709770PRTArtificial SequenceCry1B1 protein 9Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro1 5 10 15Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala20 25 30Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser35 40 45Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn50 55 60Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro65 70 75 80Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly85 90 95Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser100 105 110Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Thr Ser Asn Arg115 120 125Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser Asn His Ser Ala Gln130 135 140Met Asp Leu Leu Pro Asp Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala145 150 155 160Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Ala Ser Thr Val Gln Thr165 170 175Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Val Leu Gly Val Pro Phe180 185 190Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp195 200 205Pro Arg Gly Arg Asp Gln Trp Glu Ile Phe Leu Glu His Val Glu Gln210 215 220Leu Ile Asn Gln Gln Ile Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala225 230 235 240Arg Leu Gln Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gln Gln Ser Leu245 250 255Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg Thr Arg Ser Val Leu260 265 270His Thr Gln Tyr Ile Ala Leu Glu Leu Asp Phe Leu Asn Ala Met Pro275 280 285Leu Phe Ala Ile Arg Asn Gln Glu Val Pro Leu Leu Met Val Tyr Ala290 295 300Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe305 310 315 320Gly Ser Glu Phe Gly Leu Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu325 330 335Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser Asp Tyr Cys Val Glu Trp340 345 350Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr Asn Ala Ala Ser Trp355 360 365Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr Leu Gly Val Leu Asp370 375 380Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn385 390 395 400Thr Ser Ala Gln Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Ala405 410 415Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro420 425 430Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile Arg Ser Pro His Leu Leu435 440 445Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser Ala Ser Ser Arg Trp Ser450 455 460Asn Thr Arg His Met Thr Tyr Trp Arg Gly His Thr Ile Gln Ser Arg465 470 475 480Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr485 490 495Ser Ile Asn Pro Val Thr Leu Arg Phe Ala Ser Arg Asp Val Tyr Arg500 505 510Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly Ile Tyr Leu Glu Pro515 520 525Ile His Gly Val Pro Thr Val Arg Phe Asn Phe Thr Asn Pro Gln Asn530 535 540Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gln Pro Tyr Glu Ser Pro545 550 555 560Gly Leu Gln Leu Lys Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr565 570 575Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly580 585 590Ile Ile Leu Gln Ser Arg Val Asn Val Pro Val Tyr Ser Trp Thr His595 600 605Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pro Asn Arg Ile Thr Gln610 615 620Ile Pro Met Val Lys Ala Ser Glu Leu Pro Gln Gly Thr Thr Val Val625 630 635 640Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr645 650 655Gly Gly Phe Gly Pro Ile Arg Val Thr Val Asn Gly Pro Leu Thr Gln660 665 670Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe675 680 685Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn Phe Arg Phe Leu Arg690 695 700Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly Asn Phe Val Arg Arg705 710 715 720Ala Phe Thr Thr Pro Phe Thr Phe Thr Gln Ile Gln Asp Ile Ile Arg725 730 735Thr Ser Ile Gln Gly Leu Ser Gly Asn Gly Glu Val Tyr Ile Asp Lys740 745 750Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu755 760 765Glu Arg770101947DNAArtificial SequenceOptimized cry1B2 coding sequence 10atg gct act tcg aac aga aag aac gag aac gag atc atc aac gct gtt 48Met Ala Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val1 5 10 15tct aac cat tct gct cag atg gat ctt ctt cct gat gct aga atc gag 96Ser Asn His Ser Ala Gln Met Asp Leu Leu Pro Asp Ala Arg Ile Glu20 25 30gat tct ctt tgt atc gct gag gga aac aac atc gat cct ttc gtt tct 144Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp Pro Phe Val Ser35 40 45gct tct act gtt cag act ggt atc aac atc gct gga aga att ctt gga 192Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly50 55 60gtt ctt gga gtt cct ttc gct gga cag ctt gct tct ttc tac tct ttc 240Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe65 70 75 80ctt gtt gga gag ctt tgg cct aga gga aga gat cag tgg gag atc ttc 288Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gln Trp Glu Ile Phe85 90 95ctt gag cat gtt gag cag ctt atc aac cag cag atc act gag aac gct 336Leu Glu His Val Glu Gln Leu Ile Asn Gln Gln Ile Thr Glu Asn Ala100 105 110aga aac act gct ctt gct aga ctt cag gga ctt gga gat tct ttc aga 384Arg Asn Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp Ser Phe Arg115 120 125gct tac cag cag tct ctt gag gac tgg ctt gag aac aga gat gat gct 432Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala130 135 140aga act aga tct gtt ctt cat act cag tac atc gct ctt gag ctt gat 480Arg Thr Arg Ser Val Leu His Thr Gln Tyr Ile Ala Leu Glu Leu Asp145 150 155 160ttc ctt aac gct atg cct ctt ttc gct atc aga aac cag gag gtt cct 528Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn Gln Glu Val Pro165 170 175ctt ctt atg gtt tac gct cag gct gct aac ctt cat ctt ctt ctt ctt 576Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu180 185 190aga gat gct tct ctt ttc gga tct gag ttc gga ctt act tct cag gag 624Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gln Glu195 200 205atc cag aga tat tac gag aga cag gtt gag aga act aga gat tac tct 672Ile Gln Arg Tyr Tyr Glu Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser210 215 220gat tac tgt gtt gag tgg tac aac act gga ctt aac tct ctt aga gga 720Asp Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly225 230 235 240act aac gct gct tct tgg gtt aga tac aac cag ttc aga aga gat ctt 768Thr Asn Ala Ala Ser Trp Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu245 250 255act ctt gga gtt ctt gat ctt gtt gct ctt ttc cct tct tac gac act 816Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr260 265 270aga act tac cct atc aac act tct gct cag ctt act aga gag gtt tac 864Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr Arg Glu Val Tyr275 280 285act gat gct atc gga gct act gga gtt aac atg gct tct atg aac tgg 912Thr Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp290 295 300tac aac aac aac gct cct tct ttc tct gct atc gag gct gct gct atc 960Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile305 310 315 320aga tct cct cat ctt ctt gat ttc ctt gag cag ctt act atc ttc tct 1008Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser325 330 335gct tct tct aga tgg tct aac act aga cac atg act tac tgg aga gga 1056Ala Ser Ser Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly340 345 350cat acc atc cag tct aga cct atc gga gga gga ctt aac act tct act 1104His Thr Ile Gln Ser Arg Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr355 360 365cat gga gct act aac act tct atc aac cct gtt act ctt aga ttc gct 1152His Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Arg Phe Ala370 375 380tct aga gat gtt tac aga act gag tct tac gct gga gtt ctt ctt tgg 1200Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp385 390 395 400gga atc tac ctt gag cct atc cac gga gtt cct act gtt aga ttc aac 1248Gly Ile Tyr Leu Glu Pro Ile His Gly Val Pro Thr Val Arg Phe Asn405 410 415ttc act aac cct cag aac atc tct gat aga gga act gct aac tac tct 1296Phe Thr Asn Pro Gln Asn Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser420 425 430cag cct tac gag tct cct gga ctt cag ctt aag gat tct gag act gag 1344Gln Pro Tyr Glu Ser Pro Gly Leu Gln Leu Lys Asp Ser Glu Thr Glu435 440 445ctt cct cct gag act act gag aga cct aac tac gag tct tac tct cat 1392Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His450 455 460aga ctt tct cat atc gga atc atc ctt cag tct aga gtt aac gtt cct 1440Arg Leu Ser His Ile Gly Ile Ile Leu Gln Ser Arg Val Asn Val Pro465 470 475 480gtt tac tct tgg act cat aga tct gct gat aga act aac act atc gga 1488Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly485 490 495cct aac aga atc act cag atc cct atg gtt aag gct tct gag ctt cct 1536Pro Asn Arg Ile Thr Gln Ile Pro Met Val Lys Ala Ser Glu Leu Pro500 505 510cag gga act act gtt gtt aga gga cct gga ttc act gga gga gat atc 1584Gln Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile515 520 525ctt aga aga act aac act gga gga ttc gga cct atc aga gtt act gtt 1632Leu Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro Ile Arg Val Thr Val530 535 540aac gga cct ctt act cag aga tac aga atc gga ttc aga tac gct tct 1680Asn Gly Pro Leu Thr Gln Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser545 550 555 560act gtt gat ttc gat ttc ttc gtt tct aga gga gga act act gtt aac 1728Thr Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn565 570 575aac ttc aga ttc ctt aga act atg aac tct gga gat gag ctt aag tac 1776Asn Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr580 585 590gga aac ttc gtt aga aga gct ttc act act cct ttc act ttc act cag 1824Gly Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gln595 600 605atc cag gat atc atc aga act tct atc cag gga ctt tct gga aac gga 1872Ile Gln Asp Ile Ile Arg Thr Ser Ile Gln Gly Leu Ser Gly Asn Gly610 615 620gag gtt tac atc gat aaa atc gag atc atc cct gtt act gct act ttc 1920Glu Val Tyr Ile Asp Lys Ile Glu Ile Ile Pro Val Thr Ala Thr Phe625 630 635 640gag gct gag tac gat tta gag aga tga 1947Glu Ala Glu Tyr Asp Leu Glu Arg64511648PRTArtificial SequenceCry1B2 protein 11Met Ala Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val1 5 10 15Ser Asn His Ser Ala Gln Met Asp Leu Leu Pro Asp Ala Arg Ile Glu20 25 30Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp Pro Phe Val Ser35 40 45Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly50 55 60Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe65 70 75 80Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gln Trp Glu Ile Phe85 90 95Leu Glu His Val Glu Gln Leu Ile Asn Gln Gln Ile Thr Glu Asn Ala100 105 110Arg Asn Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp Ser Phe Arg115 120 125Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala130 135 140Arg Thr Arg Ser Val Leu His Thr Gln Tyr Ile Ala Leu Glu Leu Asp145 150 155 160Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn Gln Glu Val Pro165 170 175Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu180 185 190Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gln Glu195 200 205Ile Gln Arg Tyr Tyr Glu Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser210 215 220Asp Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly225 230 235 240Thr Asn Ala Ala Ser Trp Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu245 250 255Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr260 265 270Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr Arg Glu Val Tyr275 280 285Thr Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp290 295 300Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile305 310 315 320Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser325 330 335Ala Ser Ser Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly340 345 350His Thr Ile Gln Ser Arg Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr355 360 365His Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Arg Phe Ala370 375 380Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp385 390 395 400Gly Ile Tyr Leu Glu Pro Ile His Gly Val Pro Thr Val Arg Phe Asn405 410 415Phe Thr Asn Pro Gln Asn Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser420 425 430Gln Pro Tyr Glu Ser Pro Gly Leu Gln Leu Lys Asp Ser Glu Thr Glu435 440 445Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His450 455 460Arg Leu Ser His Ile Gly Ile Ile Leu Gln Ser Arg Val Asn Val Pro465 470 475 480Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly485 490 495Pro Asn Arg Ile Thr Gln Ile Pro Met Val Lys Ala Ser Glu Leu Pro500 505 510Gln Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile515 520 525Leu Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro Ile Arg Val Thr Val530 535 540Asn Gly Pro Leu Thr Gln Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser545 550 555 560Thr Val Asp Phe Asp Phe

Phe Val Ser Arg Gly Gly Thr Thr Val Asn565 570 575Asn Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr580 585 590Gly Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gln595 600 605Ile Gln Asp Ile Ile Arg Thr Ser Ile Gln Gly Leu Ser Gly Asn Gly610 615 620Glu Val Tyr Ile Asp Lys Ile Glu Ile Ile Pro Val Thr Ala Thr Phe625 630 635 640Glu Ala Glu Tyr Asp Leu Glu Arg645122181DNAArtificial SequenceOptimized cry1D1 coding sequence 12atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro1 5 10 15gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala20 25 30gct ttc cct act act aag aag gct aac gat ttc tct act ctt cct tct 144Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser35 40 45aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac 192Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn50 55 60aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct 240Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro65 70 75 80act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga 288Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly85 90 95ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct 336Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser100 105 110ctt gga aac gtt tct aac gga gga aga atc aga tgt gag atc aac aac 384Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Ile Asn Asn115 120 125cag aac cag tgt gtt cct tac aac tgt ctt tct aac cct aag gag atc 432Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Ser Asn Pro Lys Glu Ile130 135 140atc ctt gga gag gag aga ctt gag act gga aac act gtt gct gat atc 480Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn Thr Val Ala Asp Ile145 150 155 160tct ctt gga ctt atc aac ttc ctt tac tct aac ttc gtt cct gga ggt 528Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser Asn Phe Val Pro Gly Gly165 170 175gga ttc atc gtt gga ctt ctt gag ctt atc tgg gga ttc atc gga cct 576Gly Phe Ile Val Gly Leu Leu Glu Leu Ile Trp Gly Phe Ile Gly Pro180 185 190tct cag tgg gat atc ttc ctt gct cag atc gag cag ctt atc tct cag 624Ser Gln Trp Asp Ile Phe Leu Ala Gln Ile Glu Gln Leu Ile Ser Gln195 200 205aga atc gag gag ttc gct aga aac cag gct atc tct aga ctt gag gga 672Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly210 215 220ctt tct aat ctt tac aaa gtt tac gtt aga gct ttc tct gac tgg gag 720Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg Ala Phe Ser Asp Trp Glu225 230 235 240aag gat cct act aac cct gct ctt aga gag gag atg aga atc cag ttc 768Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe245 250 255aac gat atg aac tct gct ctt atc act gct atc cct ctt ttc aga gtt 816Asn Asp Met Asn Ser Ala Leu Ile Thr Ala Ile Pro Leu Phe Arg Val260 265 270cag aac tac gag gtt gct ctt ctt tct gtt tac gtt cag gct gct aac 864Gln Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr Val Gln Ala Ala Asn275 280 285ctt cat ctt tct atc ctt aga gat gtt tct gtt ttc gga gag aga tgg 912Leu His Leu Ser Ile Leu Arg Asp Val Ser Val Phe Gly Glu Arg Trp290 295 300gga tac gat act gct act atc aac aac aga tac tct gat ctt act tct 960Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg Tyr Ser Asp Leu Thr Ser305 310 315 320ctt atc cat gtt tac act aac cat tgt gtt gat act tac aac cag gga 1008Leu Ile His Val Tyr Thr Asn His Cys Val Asp Thr Tyr Asn Gln Gly325 330 335ctt aga aga ctt gag gga aga ttc ctt tct gac tgg atc gtt tac aac 1056Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser Asp Trp Ile Val Tyr Asn340 345 350aga ttc aga aga cag ctt act atc tct gtt ctt gat atc gtt gct ttc 1104Arg Phe Arg Arg Gln Leu Thr Ile Ser Val Leu Asp Ile Val Ala Phe355 360 365ttc cct aac tac gat atc aga act tac cct atc cag act gct act cag 1152Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Gln Thr Ala Thr Gln370 375 380ctt act aga gag gtt tac ctt gat ctt cct ttc atc aac gag aac ctt 1200Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro Phe Ile Asn Glu Asn Leu385 390 395 400tct cct gct gct tct tac cct act ttc tct gct gct gag tct gct atc 1248Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala Ala Glu Ser Ala Ile405 410 415atc aga tct cct cat ctt gtt gat ttc ctt aac tct ttc act atc tac 1296Ile Arg Ser Pro His Leu Val Asp Phe Leu Asn Ser Phe Thr Ile Tyr420 425 430act gat tct ctt gct aga tac gct tac tgg gga gga cat ctt gtt aac 1344Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp Gly Gly His Leu Val Asn435 440 445tct ttc aga act gga act aca act aac ctt atc aga tct cct ctt tac 1392Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu Ile Arg Ser Pro Leu Tyr450 455 460gga aga gag gga aac act gag aga cct gtt act atc act gct tct cct 1440Gly Arg Glu Gly Asn Thr Glu Arg Pro Val Thr Ile Thr Ala Ser Pro465 470 475 480tct gtt cct atc ttc aga act ctt tct tac atc act gga ctt gat aac 1488Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr Ile Thr Gly Leu Asp Asn485 490 495tct aac cct gtt gct gga atc gag gga gtt gag ttc cag aac act atc 1536Ser Asn Pro Val Ala Gly Ile Glu Gly Val Glu Phe Gln Asn Thr Ile500 505 510tct aga tct atc tac aga aag tct gga cct atc gat tct ttc tct gag 1584Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro Ile Asp Ser Phe Ser Glu515 520 525ctt cct cct cag gat gct tct gtt tct cct gct atc gga tac tct cat 1632Leu Pro Pro Gln Asp Ala Ser Val Ser Pro Ala Ile Gly Tyr Ser His530 535 540aga ctt tgt cat gct act ttc ctt gag aga atc tct gga cct aga atc 1680Arg Leu Cys His Ala Thr Phe Leu Glu Arg Ile Ser Gly Pro Arg Ile545 550 555 560gct gga act gtt ttc tct tgg act cat aga tct gct tct cct act aac 1728Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser Ala Ser Pro Thr Asn565 570 575gag gtt tct cct tct aga atc act cag atc cct tgg gtt aag gct cat 1776Glu Val Ser Pro Ser Arg Ile Thr Gln Ile Pro Trp Val Lys Ala His580 585 590act ctt gct tct gga gct tct gtt atc aag gga cct gga ttc act gga 1824Thr Leu Ala Ser Gly Ala Ser Val Ile Lys Gly Pro Gly Phe Thr Gly595 600 605gga gat atc ctt act aga aac tct atg gga gag ctt gga act ctt aga 1872Gly Asp Ile Leu Thr Arg Asn Ser Met Gly Glu Leu Gly Thr Leu Arg610 615 620gtt act ttc act gga aga ctt cct cag tct tac tac atc aga ttc aga 1920Val Thr Phe Thr Gly Arg Leu Pro Gln Ser Tyr Tyr Ile Arg Phe Arg625 630 635 640tac gct tct gtt gct aac aga tct gga act ttc aga tac tct cag cct 1968Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr Phe Arg Tyr Ser Gln Pro645 650 655cct tct tac gga atc tct ttc cct aag act atg gat gct gga gag cct 2016Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr Met Asp Ala Gly Glu Pro660 665 670ctt act tct aga tct ttc gct cat aca act ctt ttc act cct atc act 2064Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu Phe Thr Pro Ile Thr675 680 685ttc tct aga gct cag gag gag ttc gat cta tac atc cag tct gga gtt 2112Phe Ser Arg Ala Gln Glu Glu Phe Asp Leu Tyr Ile Gln Ser Gly Val690 695 700tac atc gat aga atc gag ttc atc cct gtt act gct act ttc gag gct 2160Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Thr Ala Thr Phe Glu Ala705 710 715 720gag tac gat tta gag aga tga 2181Glu Tyr Asp Leu Glu Arg72513726PRTArtificial SequenceCry1D1 protein 13Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro1 5 10 15Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala20 25 30Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser35 40 45Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn50 55 60Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro65 70 75 80Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly85 90 95Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser100 105 110Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Ile Asn Asn115 120 125Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Ser Asn Pro Lys Glu Ile130 135 140Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn Thr Val Ala Asp Ile145 150 155 160Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser Asn Phe Val Pro Gly Gly165 170 175Gly Phe Ile Val Gly Leu Leu Glu Leu Ile Trp Gly Phe Ile Gly Pro180 185 190Ser Gln Trp Asp Ile Phe Leu Ala Gln Ile Glu Gln Leu Ile Ser Gln195 200 205Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly210 215 220Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg Ala Phe Ser Asp Trp Glu225 230 235 240Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe245 250 255Asn Asp Met Asn Ser Ala Leu Ile Thr Ala Ile Pro Leu Phe Arg Val260 265 270Gln Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr Val Gln Ala Ala Asn275 280 285Leu His Leu Ser Ile Leu Arg Asp Val Ser Val Phe Gly Glu Arg Trp290 295 300Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg Tyr Ser Asp Leu Thr Ser305 310 315 320Leu Ile His Val Tyr Thr Asn His Cys Val Asp Thr Tyr Asn Gln Gly325 330 335Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser Asp Trp Ile Val Tyr Asn340 345 350Arg Phe Arg Arg Gln Leu Thr Ile Ser Val Leu Asp Ile Val Ala Phe355 360 365Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Gln Thr Ala Thr Gln370 375 380Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro Phe Ile Asn Glu Asn Leu385 390 395 400Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala Ala Glu Ser Ala Ile405 410 415Ile Arg Ser Pro His Leu Val Asp Phe Leu Asn Ser Phe Thr Ile Tyr420 425 430Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp Gly Gly His Leu Val Asn435 440 445Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu Ile Arg Ser Pro Leu Tyr450 455 460Gly Arg Glu Gly Asn Thr Glu Arg Pro Val Thr Ile Thr Ala Ser Pro465 470 475 480Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr Ile Thr Gly Leu Asp Asn485 490 495Ser Asn Pro Val Ala Gly Ile Glu Gly Val Glu Phe Gln Asn Thr Ile500 505 510Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro Ile Asp Ser Phe Ser Glu515 520 525Leu Pro Pro Gln Asp Ala Ser Val Ser Pro Ala Ile Gly Tyr Ser His530 535 540Arg Leu Cys His Ala Thr Phe Leu Glu Arg Ile Ser Gly Pro Arg Ile545 550 555 560Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser Ala Ser Pro Thr Asn565 570 575Glu Val Ser Pro Ser Arg Ile Thr Gln Ile Pro Trp Val Lys Ala His580 585 590Thr Leu Ala Ser Gly Ala Ser Val Ile Lys Gly Pro Gly Phe Thr Gly595 600 605Gly Asp Ile Leu Thr Arg Asn Ser Met Gly Glu Leu Gly Thr Leu Arg610 615 620Val Thr Phe Thr Gly Arg Leu Pro Gln Ser Tyr Tyr Ile Arg Phe Arg625 630 635 640Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr Phe Arg Tyr Ser Gln Pro645 650 655Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr Met Asp Ala Gly Glu Pro660 665 670Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu Phe Thr Pro Ile Thr675 680 685Phe Ser Arg Ala Gln Glu Glu Phe Asp Leu Tyr Ile Gln Ser Gly Val690 695 700Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Thr Ala Thr Phe Glu Ala705 710 715 720Glu Tyr Asp Leu Glu Arg725141815DNAArtificial SequenceOptimized cry1D2 coding sequence 14atg gct gag atc aac aac cag aac cag tgt gtt cct tac aac tgt ctt 48Met Ala Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu1 5 10 15tct aac cct aag gag atc atc ctt gga gag gag aga ctt gag act gga 96Ser Asn Pro Lys Glu Ile Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly20 25 30aac act gtt gct gat atc tct ctt gga ctt atc aac ttc ctt tac tct 144Asn Thr Val Ala Asp Ile Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser35 40 45aac ttc gtt cct gga ggt gga ttc atc gtt gga ctt ctt gag ctt atc 192Asn Phe Val Pro Gly Gly Gly Phe Ile Val Gly Leu Leu Glu Leu Ile50 55 60tgg gga ttc atc gga cct tct cag tgg gat atc ttc ctt gct cag atc 240Trp Gly Phe Ile Gly Pro Ser Gln Trp Asp Ile Phe Leu Ala Gln Ile65 70 75 80gag cag ctt atc tct cag aga atc gag gag ttc gct aga aac cag gct 288Glu Gln Leu Ile Ser Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala85 90 95atc tct aga ctt gag gga ctt tct aat ctt tac aaa gtt tac gtt aga 336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg100 105 110gct ttc tct gac tgg gag aag gat cct act aac cct gct ctt aga gag 384Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu115 120 125gag atg aga atc cag ttc aac gat atg aac tct gct ctt atc act gct 432Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Ile Thr Ala130 135 140atc cct ctt ttc aga gtt cag aac tac gag gtt gct ctt ctt tct gtt 480Ile Pro Leu Phe Arg Val Gln Asn Tyr Glu Val Ala Leu Leu Ser Val145 150 155 160tac gtt cag gct gct aac ctt cat ctt tct atc ctt aga gat gtt tct 528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Ile Leu Arg Asp Val Ser165 170 175gtt ttc gga gag aga tgg gga tac gat act gct act atc aac aac aga 576Val Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg180 185 190tac tct gat ctt act tct ctt atc cat gtt tac act aac cat tgt gtt 624Tyr Ser Asp Leu Thr Ser Leu Ile His Val Tyr Thr Asn His Cys Val195 200 205gat act tac aac cag gga ctt aga aga ctt gag gga aga ttc ctt tct 672Asp Thr Tyr Asn Gln Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser210 215 220gac tgg atc gtt tac aac aga ttc aga aga cag ctt act atc tct gtt 720Asp Trp Ile Val Tyr Asn Arg Phe Arg Arg Gln Leu Thr Ile Ser Val225 230 235 240ctt gat atc gtt gct ttc ttc cct aac tac gat atc aga act tac cct 768Leu Asp Ile Val Ala Phe Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro245 250 255atc cag act gct act cag ctt act aga gag gtt tac ctt gat ctt cct 816Ile Gln Thr Ala Thr Gln Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro260 265 270ttc atc aac gag aac ctt tct cct gct gct tct tac cct act ttc tct 864Phe Ile Asn Glu Asn Leu Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser275 280 285gct gct gag tct gct atc atc aga tct cct cat ctt gtt gat ttc ctt 912Ala Ala Glu Ser Ala Ile Ile Arg Ser Pro His Leu Val Asp Phe Leu290 295 300aac tct ttc act atc tac act gat tct ctt gct aga tac gct tac tgg 960Asn Ser Phe Thr Ile Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp305 310 315 320gga gga cat ctt gtt aac tct ttc aga act gga act aca act aac ctt 1008Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu325 330 335atc aga tct cct ctt tac gga aga gag gga aac act gag aga cct gtt 1056Ile Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val340 345 350act atc act gct tct cct tct gtt cct atc ttc aga act ctt tct tac 1104Thr Ile Thr Ala Ser Pro Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr355 360 365atc act gga ctt gat aac tct aac cct gtt gct gga atc gag gga gtt 1152Ile

Thr Gly Leu Asp Asn Ser Asn Pro Val Ala Gly Ile Glu Gly Val370 375 380gag ttc cag aac act atc tct aga tct atc tac aga aag tct gga cct 1200Glu Phe Gln Asn Thr Ile Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro385 390 395 400atc gat tct ttc tct gag ctt cct cct cag gat gct tct gtt tct cct 1248Ile Asp Ser Phe Ser Glu Leu Pro Pro Gln Asp Ala Ser Val Ser Pro405 410 415gct atc gga tac tct cat aga ctt tgt cat gct act ttc ctt gag aga 1296Ala Ile Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg420 425 430atc tct gga cct aga atc gct gga act gtt ttc tct tgg act cat aga 1344Ile Ser Gly Pro Arg Ile Ala Gly Thr Val Phe Ser Trp Thr His Arg435 440 445tct gct tct cct act aac gag gtt tct cct tct aga atc act cag atc 1392Ser Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg Ile Thr Gln Ile450 455 460cct tgg gtt aag gct cat act ctt gct tct gga gct tct gtt atc aag 1440Pro Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val Ile Lys465 470 475 480gga cct gga ttc act gga gga gat atc ctt act aga aac tct atg gga 1488Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr Arg Asn Ser Met Gly485 490 495gag ctt gga act ctt aga gtt act ttc act gga aga ctt cct cag tct 1536Glu Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro Gln Ser500 505 510tac tac atc aga ttc aga tac gct tct gtt gct aac aga tct gga act 1584Tyr Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr515 520 525ttc aga tac tct cag cct cct tct tac gga atc tct ttc cct aag act 1632Phe Arg Tyr Ser Gln Pro Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr530 535 540atg gat gct gga gag cct ctt act tct aga tct ttc gct cat aca act 1680Met Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr545 550 555 560ctt ttc act cct atc act ttc tct aga gct cag gag gag ttc gat cta 1728Leu Phe Thr Pro Ile Thr Phe Ser Arg Ala Gln Glu Glu Phe Asp Leu565 570 575tac atc cag tct gga gtt tac atc gat aga atc gag ttc atc cct gtt 1776Tyr Ile Gln Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val580 585 590act gct act ttc gag gct gag tac gat tta gag aga tga 1815Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg595 60015604PRTArtificial SequenceCry1D2 protein 15Met Ala Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu1 5 10 15Ser Asn Pro Lys Glu Ile Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly20 25 30Asn Thr Val Ala Asp Ile Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser35 40 45Asn Phe Val Pro Gly Gly Gly Phe Ile Val Gly Leu Leu Glu Leu Ile50 55 60Trp Gly Phe Ile Gly Pro Ser Gln Trp Asp Ile Phe Leu Ala Gln Ile65 70 75 80Glu Gln Leu Ile Ser Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala85 90 95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg100 105 110Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu115 120 125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Ile Thr Ala130 135 140Ile Pro Leu Phe Arg Val Gln Asn Tyr Glu Val Ala Leu Leu Ser Val145 150 155 160Tyr Val Gln Ala Ala Asn Leu His Leu Ser Ile Leu Arg Asp Val Ser165 170 175Val Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg180 185 190Tyr Ser Asp Leu Thr Ser Leu Ile His Val Tyr Thr Asn His Cys Val195 200 205Asp Thr Tyr Asn Gln Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser210 215 220Asp Trp Ile Val Tyr Asn Arg Phe Arg Arg Gln Leu Thr Ile Ser Val225 230 235 240Leu Asp Ile Val Ala Phe Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro245 250 255Ile Gln Thr Ala Thr Gln Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro260 265 270Phe Ile Asn Glu Asn Leu Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser275 280 285Ala Ala Glu Ser Ala Ile Ile Arg Ser Pro His Leu Val Asp Phe Leu290 295 300Asn Ser Phe Thr Ile Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp305 310 315 320Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu325 330 335Ile Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val340 345 350Thr Ile Thr Ala Ser Pro Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr355 360 365Ile Thr Gly Leu Asp Asn Ser Asn Pro Val Ala Gly Ile Glu Gly Val370 375 380Glu Phe Gln Asn Thr Ile Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro385 390 395 400Ile Asp Ser Phe Ser Glu Leu Pro Pro Gln Asp Ala Ser Val Ser Pro405 410 415Ala Ile Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg420 425 430Ile Ser Gly Pro Arg Ile Ala Gly Thr Val Phe Ser Trp Thr His Arg435 440 445Ser Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg Ile Thr Gln Ile450 455 460Pro Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val Ile Lys465 470 475 480Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr Arg Asn Ser Met Gly485 490 495Glu Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro Gln Ser500 505 510Tyr Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr515 520 525Phe Arg Tyr Ser Gln Pro Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr530 535 540Met Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr545 550 555 560Leu Phe Thr Pro Ile Thr Phe Ser Arg Ala Gln Glu Glu Phe Asp Leu565 570 575Tyr Ile Gln Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val580 585 590Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg595 60016372DNAArtificial SequenceOptimized chloroplast transit peptide 16atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro1 5 10 15gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala20 25 30gct ttc cct act act aag aag gct aac gat ttc tct act ctt cct tct 144Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser35 40 45aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac 192Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn50 55 60aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct 240Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro65 70 75 80act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga 288Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly85 90 95ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct 336Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser100 105 110ctt gga aac gtt tct aac gga gga aga atc aga tgt 372Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys115 12017124PRTArtificial SequenceChloropolast transit peptide 17Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro1 5 10 15Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala20 25 30Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser35 40 45Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn50 55 60Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro65 70 75 80Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly85 90 95Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser100 105 110Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys115 12018988DNASubterranean clover stunt virus 18ctagataatt gttattatca ataaaagaat ttttattgtt attgtgttat ttggtaattt 60atgcttataa gtaattctat gattaattgt gaattaataa gactaatgag gataataatt 120gaatttgatt aaattaactc tgcgaagcca tatgtctttc acgtgagagt cacgtgatgt 180ctccgcgaca ggctggcacg gggcttagta ttacccccgt gccgggatca gagacatttg 240actaaatgtt gacttggaat aatagccctt ggattagatg acacgtggac gctcaggatc 300tgtgatgcta gtgaagcgct taagctgaac gaatctgacg gaagagcgga caaacgcaca 360tggactatgg cccactgctt tattaaagaa gtgaatgaca gctgtctttg cttcaagacg 420aagtaaagaa tagtggaaaa cgcgttaatt gttattatca ataaaagaat ttttattgtt 480attgtgttat ttggtaattt atgcttataa gtaattctat gattaattgt gaattaataa 540gactaatgag gataataatt gaatttgatt aaattaactc tgcgaagcta tatgtctttc 600acgtgagagt cacgtgatgt ctccgcgaca ggctggcacg gggcttagta ttacccccgt 660gccgggatca gagacatttg actaaatgtt gacttggaat aatagccctt ggattagatg 720acacgtggac gctcaggatc tgtgatgcta gtgaagcgct taagctgaac gaatctgacg 780gaagagcgga caaacgcaca tggactatgg cccactgctt tattaaagaa gtgaatgaca 840gctgtctttg cttcaagacg aagtaaagaa tagtggaaaa cgcgtaaaga ataagcgtac 900tcagtacgct tcgtggcttt ataaatagtg cttcgtctta ttcttcgttg tatcatcaac 960gaagaagtta agctttgttc tgcgtttc 988191042DNASubterranean clover stunt virus 19taattaatag taattatgat taattatgag ataagagttg ttattaatgc ttatgaggaa 60taaagaatga ttaatattgt ttaattttat tccgcgaagc ggtgtgttat gtttttgttg 120gagacatcac gtgactctca cgtgatgtct ccgcgacagg ctggcacggg gcttagtatt 180acccccgtgc cgggatcaga gacatttgac taaatattga cttggaataa tagcccttgg 240attagatgac acgtggacgc tcaggatctg tgatgctagt gaagcgctta agctgaacga 300atctgacgga agagcggaca tacgcacatg gattatggcc cacatgtcta aagtgtatct 360ctttacagct atatcgatgt gacgtaagat gctttacttc gcttcgaagt aaagtaggaa 420attgctcgct aagttattct tttctgaaag aaattaattt aattctaatt aaattaaatg 480agtggcctgc agtaattaat agtaattatg attaattatg agataagagt tgttattaat 540gcttatgagg aataaagaat gattaatatt gtttaatttt attccgcgaa gcggtgtgtt 600atgtttttgt tggagacatc acgtgactct cacgtgatgt ctccgcgaca ggctggcacg 660gggcttagta ttacccccgt gccgggatca gagacatttg actaaatatt gacttggaat 720aatagccctt ggattagatg acacgtggac gctcaggatc tgtgatgcta gtgaagcgct 780taagctgaac gaatctgacg gaagagcgga catacgcaca tggattatgg cccacatgtc 840taaagtgtat ctctttacag ctatatcgat gtgacgtaag atgctttact tcgcttcgaa 900gtaaagtagg aaattgctcg ctaagttatt cttttctgaa agaaattaat ttaattctaa 960attaaattaa atgagtggct ataaatagtg tcgatgctac ctcacatcgt attcttcttc 1020gcatcgtctg ttctggtttt aa 10422028DNAArtificial SequenceCry1B gene primer 20tacttcgaac agaaagaacg agaacgag 282124DNAArtificial SequenceCry1B gene primer 21gtccagcgaa aggaactcca agaa 242221DNAArtificial SequenceCry1C gene primer 22aaccttgagg gacttggaaa c 212323DNAArtificial SequenceCry1C gene primer 23aagatgaggg tttctgatag cag 23

Claims

1. A chimeric gene, comprising the following operably-linked sequences: a) a coding region encoding a Cry1C protein, comprising the DNA of any one of SEQ ID Nos. 1, 3, 4 or 6, and b) a promoter region capable of directing expression in plant cells.

2. The chimeric gene of claim 1, wherein said promoter comprises the sequence of SEQ ID No. 18 or 19.

3. The chimeric gene of claim 1, further comprising a 3' polyadenylation and transcript termination region.

4. The chimeric gene of claim 3, wherein said 3' polyadenylation and transcript termination region is of the NADP-malic enzyme gene from Flaveria bidentis.

5. The chimeric gene of claim 1, further comprising the leader sequence of the tapetum specific E1 gene of Oryza sativa between the promoter and the coding region.

6. A DNA comprising the chimeric gene of claim 1, further comprising a second chimeric gene, comprising the following operably-linked sequences: a) a second coding region encoding a Cry1B protein comprising the DNA of SEQ ID No. 8 or 10, and b) a second promoter region capable of directing expression in plant cells.

7. A DNA comprising the chimeric gene of claim 1, further comprising a second chimeric gene comprising the following operably-linked sequences: a) a coding region encoding a Cry1D protein comprising the DNA of SEQ ID No. 12 or 14, and b) a promoter region capable of directing expression in plant cells.

8. The DNA of claim 6, wherein said second promoter region comprises the sequence of SEQ ID No. 18 or 19 and is different from said first promoter region.

9. The DNA of claim 6, wherein said second chimeric gene further comprises a 3' polyadenylation and transcript termination region.

10. The DNA of claim 9, wherein said 3' polyadenylation and transcript termination region is of the NADP-malic enzyme gene from Flaveria bidentis.

11. The DNA of claim 6, wherein said second chimeric gene further comprises the leader sequence of the tapetum specific E1 gene of Oryza sativa between the promoter and the coding region.

12. The DNA of claim 6, further comprising a third chimeric gene, comprising the following operably-linked sequences: a) a coding region encoding a Cry1D protein comprising the DNA of SEQ ID No. 12 or 14, and b) a promoter region capable of directing expression in plants.

13. A transgenic plant cell, comprising the gene of claim 1 stably incorporated in its genome.

14. A plant comprising the gene of claim 1 stably incorporated in its genome.

15. The plant of claim 14, which is a Brassica species plant or plant cell.

16. The plant of claim 15, wherein said plant is of the species Brassica oleraceae, Brassica napus, Brassica rapa, Brassica juncea or Brassica carinata.

17. The plant of claim 16, wherein said species is cabbage or cauliflower.

18. (canceled)

19. A method for controlling insects, comprising: planting or sowing in a field, plants comprising the chimeric gene of claim 1.

20. A chimeric gene comprising the following operably-linked sequences: a) a first fragment of a coding sequence encoding an insecticidal protein, b) a plant intron sequence, c) a second fragment of said coding sequence, d) a promoter region capable of directing expression in plant cells.

21. A method of controlling insects in Brassica species plants, comprising: expressing the chimeric genes claim 1 in plants.

22. A method of producing plants or seeds resistant to insects, comprising the steps of: a) obtaining a plant transformed with the gene of claim 1, and b) selecting progeny of said plant or seeds thereof, containing said gene.

23. A microorganism comprising the chimeric gene of claim 1.

24. The microorganism of claim 23, wherein said microorganism is of the genus Escherichia, Bacillus, or Agrobacterium.

25. (canceled)

26. (canceled)

27. (canceled)

28. The chimeric gene of claim 1, wherein the Cry1C protein is a variant comprising the sequence of SEQ ID No. 2 from amino acid position 29 to amino acid position 627, but wherein one, some or all of the following amino acids at the following positions compared to the positions in SEQ ID No. 2 are changed: the amino acid at position 125 is Alanine, the amino acid at position 184 is Valine, the amino acid at position 295 is Arginine, the amino acid at position 454 is Aspartic acid, or the amino acid at position 593 is Arginine.

29. The DNA of claim 6, wherein the Cry1B protein is a variant comprising the sequence of SEQ ID No. 11 from amino acid position 31 to 648, but wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine or the amino acid at position 353 in SEQ ID No. 11 is Arginine, or a protein wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine and the amino acid at position 353 in SEQ ID No. 11 is Arginine.

30. (canceled)

31. A transgenic plant cell, comprising the gene of claim 28 stably incorporated in its genome.

32. A DNA encoding an optimized chloroplast transit peptide, comprising the sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position 371.

33. The chimeric gene of claim 20, wherein the plant intron is the second intron of the ST-LS1 gene of Solanum tuberosum.

34. A transgenic plant cell comprising the DNA of claim 6 stably incorporated in its genome.

35. A plant comprising the DNA of claim 6 stably incorporated in its genome.

36. The plant of claim 35, wherein said plant is of the species Brassica oleraceae, Brassica napus, Brassica rape, Brassica juncea, or Brassica carinata.

37. The plant of claim 36, wherein said plant is cabbage or cauliflower.

38. A method for controlling insects comprising planting or sowing in a field, plants comprising the DNA of claim 6.

39. A method of controlling insects in Brassica species plants, comprising expressing the DNA of claim 6 in said plants.

40. A method of producing plants or seeds resistant to insects, comprising the steps of: a) obtaining a plant transformed with the DNA of claim 6, and b) selecting progeny of said plant or seeds thereof, containing said DNA.

41. A microorganism comprising the DNA of claim 6.

42. The microorganism of claim 41, wherein said microorganism is of the genus Escherichia, Bacillus, or Agrobacterium.