Expression of Cry3B insecticidal protein in plants
AssigneeMonsanto Technology LLC
InventorCharles P. Romano
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Abstract
The present invention discloses methods and compositions comprising a group of novel expression cassettes which provide significantly improved levels of accumulation of Coleopteran inhibitory Cry3B and Cry3B variant amino acid sequences when these are expressed in plants. The preferred embodiments of the invention provide at least up to ten fold higher levels of insect controlling protein relative to the highest levels obtained using prior compositions. In particular, transgenic maize expressing higher levels of a protein designed to exhibit increased toxicity toward Coleopteran pests deliver superior levels of insect protection and are less likely to sponsor development of populations of target insects that are resistant to the insecticidally active protein.
Claims47
A plant comprising a polynucleotide sequence comprising an expression cassette containing a linear arrangement of genetic sequences which function together in plant cells to achieve improved expression of at least an insecticidal portion of a protein or amino acid sequence variant thereof from a nucleic acid coding sequence in said plant cells, said protein or variant being derived from a Bacillus thuringiensis Cry3 δ-endotoxin, said variant exhibiting an insecticidal activity toxic to a Coleopteran insect pest which is at least equivalent to said Cry3, wherein said genetic sequences comprise a promoter operably linked in linear sequence to an untranslated leader, an intron, said nucleic acid coding sequence, and a transcription termination and polyadenylation sequence.
A plant comprising a polynucleotide sequence comprising an expression cassette containing a linear arrangement of genetic sequences which function together in plant cells to achieve improved expression of a Bacillus thuringiensis Cry3 insecticidal δ-endotoxin protein or amino acid sequence variant thereof toxic to a Coleopteran insect pest from a nucleic acid coding sequence in said plant cells, said variant exhibiting an insecticidal activity at least equivalent to said Cry3, wherein said genetic sequences comprise a promoter operably linked in linear sequence to an untranslated leader, an intron, a nucleotide sequence encoding a plastid or chloroplast targeting peptide linked in frame and adjacent to said nucleic acid coding sequence, and a transcription termination and polyadenylation sequence.
A plant comprising a polynucleotide sequence comprising an expression cassette containing a linear arrangement of genetic sequences which function together in plant cells to achieve expression from a nucleic acid sequence encoding an insecticidal Bacillus thuringiensis Cry3 δ-endotoxin protein or amino acid sequence variant thereof toxic to a Coleopteran insect pest feeding on said plant, said variant exhibiting an insecticidal activity at least equivalent to said Cry3, said genetic sequences comprising a promoter operably linked in linear sequence to an untranslated leader, an intron, said nucleic acid coding sequence and a transcription termination and polyadenylation sequence, wherein said expression of said protein or variant in plant cells of said plant is improved to levels of protein or variant sufficient to protect said plant from Coleopteran pest infestation and sufficient to delay the onset of insect resistance to said protein or variant.
A method for producing a transgenic plant comprising the steps of:
- a) introducing into the DNA of a plant cell or plant tissue a polynucleotide sequence comprising an expression cassette containing a linear arrangement of genetic sequences which function together in plant cells to achieve expression from a nucleic acid sequence encoding an insecticidal Bacillus thuringiensis Cry3 δ-endotoxin protein or amino acid sequence variant thereof toxic to a Coleopteran insect pest feeding on said plant, said variant exhibiting an insecticidal activity at least equivalent to said Cry3, said genetic sequences comprising a promoter operably linked in linear sequence to an untranslated leader, an intron, said nucleic acid coding sequence, and a transcription termination and polyadenylation sequence;
- b) growing said plant cell or plant tissue in selective media to obtain a transgenic plant containing said polynucleotide sequence heritably incorporated into said DNA;
- and
- c) selecting a plant expressing said protein or variant;
- wherein said selected plant exhibits improved expression of said protein or variant in said transgenic plant, said improved expression being improved to levels of protein or variant sufficient to protect said plant from Coleopteran pest infestation and sufficient to delay the onset of insect resistance to said protein or variant when produced in said transgenic plant.
An isolated and purified polynucleotide sequence comprising an expression cassette containing a linear arrangement of genetic sequences which function together in plant cells to achieve improved expression of at least an insecticidal portion of a protein or amino acid sequence variant thereof from a nucleic acid coding sequence in said plant cells, said protein or variant being derived from a Bacillus thuringiensis Cry3 δ-endotoxin, said variant exhibiting an insecticidal activity toxic to a Coleopteran insect pest which is at least equivalent to said Cry3, wherein said genetic sequences comprise a promoter operably linked in linear sequence to an untranslated leader, an intron, said nucleic acid coding sequence, and a transcription termination and polyadenylation sequence, and wherein said expression cassette improves the expression of the protein or variant in a transgenic plant and, improves the number of transgenic events observed to express the protein or variant above a threshold level when said polynucleotide sequence is used for generating transformed plants.
An isolated and purified polynucleotide sequence comprising one or more expression cassettes, each cassette comprising genetic sequence elements which function in plant cells to express a desired Bacillus thuringiensis δ-endotoxin insecticidal protein, chimera, fusion, or variant thereof from a nucleic acid coding sequence, wherein said coding sequence is linked upstream to a promoter sequence element, an untranslated leader sequence element, an intron sequence element, and said coding sequence is linked downstream to a transcription termination and polyadenylation sequence element, wherein said expression cassette improves the expression of the protein or variant in a transgenic plant and improves the percentage of transgenic events observed to express the desired protein above a threshold level when using said polynucleotide sequence for generating transformed plants.
A method for controlling Coleopteran insect infestation in a field of crop plants comprising providing a transgenic plant on which said Coleopteran insect feeds, said transgenic plant comprising a polynucleotide sequence comprising an expression cassette containing a linear arrangement of genetic sequences which function together in plant cells to achieve improved expression of at least an insecticidal portion of a protein or amino acid sequence variant thereof from a nucleic acid coding sequence in said plant cells, said protein or variant being derived from a Bacillus thuringiensis Cry3 δ-endotoxin, said variant exhibiting an insecticidal activity toxic to a Coleopteran insect pest which is at least equivalent to said Cry3, wherein said genetic sequences comprise a promoter operably linked in linear sequence to an untranslated leader, an intron, said nucleic acid coding sequence, and a transcription termination and polyadenylation sequence.
Dependencies are inferred from the claim text.