US20100235951A1

US20100235951A1 - Novel genes encoding insecticidal proteins

Info

Publication number: US20100235951A1
Application number: US12/293,772
Authority: US
Inventors: Jeroen Van Rie; Frank Meulewaeter; Gerben Van Eldik
Original assignee: Bayer Bioscience NV
Current assignee: Bayer CropScience NV
Priority date: 2006-03-21
Filing date: 2007-03-16
Publication date: 2010-09-16
Also published as: EA200802018A1; US20170107534A1; AR059995A1; US11060103B2; EA019029B1; AU2007228981A1; CA2646471C; CN101405296B; CN101405296A; PL1999141T3; AU2007228981B2; EP1999141A2; EP1999141B1; WO2007107302A2; WO2007107302A3; CA2646471A1

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

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

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™ potatoes comprising a Cry3A Coleopteran-active gene were briefly commercialized in Northern America, but have been withdrawn from the market in 2001.
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.
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

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

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.
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.
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).
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.
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.
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 (×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).
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.
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.
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.
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× Denhardt's reagent and 0.1% SDS at 42° C., or 1 to 2 hours in 6× SSC, 2× Denhardt′s reagent and 0.1% SDS at 68 ° 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× SSC, 0.1% SDS, followed by 2 washes of 30 minutes each at 68° C. in 0.5× 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° 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.
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.
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.
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.
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.
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.
“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.
“(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.
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.
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.
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.
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 (Höfte 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
“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.
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.
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), Höfte 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:
http://www.lifesci.sussex.ac.uk/home/Neil Crickmore/Bt/index.html).
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.
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.
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.
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.
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).
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 Klösgen et al. (1989), Klösgen 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.
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.
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.
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.
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.
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.
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.
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 Schünmann 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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The enclosed sequence listing referred to in the Examples, the Claims and the Description is as follows:
Sequence Listing:
SEQ ID No.1: optimized cry1C1 coding sequence comprising an intron at position 672
SEQ ID No.2: amino acid sequence of the Cry1C1 protein encoded by SEQ ID No. 1
SEQ ID No.3: optimized cry1C2 coding sequence, comprising an intron at position 489
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
SEQ ID No.5: Cry1C3 protein encoded by SEQ ID No. 4
SEQ ID No.6: optimized cry1C4 coding sequence, comprising the sequences of SEQ ID
No. 3 and SEQ ID No. 16, encoding a fusion protein with a transit peptide
SEQ ID No.7: Cry1C4 protein encoded by SEQ ID No. 6
SEQ ID No.8: optimized cry1B1 coding sequence, including a transit peptide coding sequence
SEQ ID No.9: Cry1B1 protein encoded by the sequence of SEQ ID No. 8
SEQ ID No.10: optimized cry1B2 coding sequence
SEQ ID No.11. Cry1B2 protein encoded by the sequence of SEQ ID No. 10
SEQ ID No.12: optimized cry1D1 coding sequence, including a transit peptide coding sequence
SEQ ID No.13: Cry1D1 protein encoded by the sequence of SEQ ID No.12
SEQ ID No.14: optimized cry1D2 coding sequence
SEQ ID No.15: Cry1D2 protein encoded by the sequence of SEQ ID No. 14
SEQ ID No.16: coding sequence encoding an optimized chloroplast transit peptide
SEQ ID No.17: chloroplast transit peptide encoded by the sequence of SEQ ID No. 16
SEQ ID No.18: duplicated S7 subterranean clover stunt virus promoter sequence (S7S7)
SEQ ID No.19: duplicated S4 subterranean clover stunt virus promoter sequence (S4S4)
SEQ ID No. 20: cry1B gene primer P1B227
SEQ ID No. 21: cry1B gene primer P1B228
SEQ ID No. 22: cry1C gene primer P1C247
SEQ ID No. 23: cry1C gene primer P1C252

EXAMPLES

1.Construction of Chimeric Genes and Transformation Vectors

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

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.
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° 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 1×10⁷bacteria/ml. After the co-cultivation period, the explants are washed in liquid medium with appropriate antibiotics and hormones and blotted dry on filter paper.
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.
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.
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

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.
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.
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.
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.

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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:

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.