Methods for transforming plants to express Cry2Ab δ-endotoxins targeted to the plastids
AssigneeMonsanto Technology LLC
InventorsDavid R. Corbin, Charles P. Romano
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Abstract
Disclosed is a means of controlling plant pests by a novel method of expressing Cry2Ab B. thuringiensis δ-endotoxins in plants, targeted to the plastids. The invention comprises novel nucleic acid segments encoding proteins comprising Cry2Ab B. thuringiensis δ-endotoxins. The nucleic acid segments are disclosed, as are transformation vectors containing the nucleic acid segments, plants transformed with the claimed segments, methods for transforming plants, and methods of controlling plant infestation by pests.
Claims63
A plant comprising a nucleotide sequence encoding a Cry2Ab Bacillus thuringiensis δ-endotoxin protein comprising a plant functional promoter sequence operably linked to a first polynucleotide sequence encoding a plastid transit peptide, which linked in-frame to a second polynucleotide sequence encoding said Cry2Ab Bacillus thuringiensis δ-endotoxin protein, said second polynucleotide sequence being operably linked to a plant functional 3′ end transcription termination and polyadenylation sequence, wherein expression of said nucleotide sequence in said plant yields a fusion protein comprised of an amino-terminal plastid transit peptide covalently linked to said δ-endotoxin protein, and said plastid transit peptide functions to localize said δ-endotoxin protein to a plastid.
A method of producing a first transgenic plant which exhibits increased levels of accumulation of a Cry2Ab Bacillus thuringiensis δ-endotoxin protein and decreased phytotoxic effects of expression of a nucleotide sequence encoding said Cry2Ab Bacillus thuringiensis δ-endotoxin protein when compared to a second transgenic plant, said method comprising the steps of:
- a) transforming a plant cell with a nucleotide sequence comprising in operable linkage a plant functional promoter sequence linked to a first polynucleotide sequence encoding a plastid transit peptide and a second polynucleotide sequence encoding said Cry2Ab Bacillus thuringiensis δ-endotoxin protein;
- b) regenerating said first transgenic plant from said transformed plant cell;
- and
- c) comparing the level of Cry2Ab Bacillus thuringiensis δ-endotoxin protein accumulation and plant morphology of said first transgenic plant to said second transgenic plant transformed to express a nucleic acid sequence encoding a Cry2Ab Bacillus thuringiensis δ-endotoxin protein that is not linked to a plastid transit peptide,
- wherein expression of said nucleotide sequence in said first transgenic plant yields a fusion protein comprising an amino-terminal plastid transit peptide covalently linked to said δ-endotoxin protein, and said plastid transit peptide functions to localize said δ-endotoxin protein to a plastid.
A method for increasing the frequency of morphologically normal transgenic plants obtained after transformation which express a Cry2Ab Bacillus thuringiensis δ-endotoxin protein localized to a plastid comprising the steps of:
- a) transforming plant cells with a nucleotide sequence comprising in operable linkage a plant functional promoter linked to a first polynucleotide sequence encoding a plastid transit peptide, and a second polynucleotide sequence encoding said Cry2Ab Bacillus thuringiensis δ-endotoxin protein;
- b) selecting plant cells expressing said protein;
- c) regenerating transgenic plants from said selected plant cells;
- and
- d) comparing the number of morphologically normal transgenic plants expressing said protein to the number of plants obtained after transformation which express a nucleic acid encoding a protein selected from the group consisting of a Cry2A protein which is not localized to a plastid and a Cry2A protein other than a Cry2Ab protein localized to a plastid;
- wherein expression of said nucleotide sequence in said transgenic plants yields a fusion protein comprised of an amino-terminal plastid transit peptide covalently linked to said δ-endotoxin protein, and said plastid transit peptide functions to localize said δ-endotoxin protein to a plastid.
A method of identifying a morphologically normal transgenic plant which is resistant to insect infestation comprising:
- a) obtaining a first plant containing a nucleic acid sequence comprising a plant functional promoter operably linked to a first polynucleotide sequence encoding a plastid transit peptide, which is linked in frame to a second polynucleotide sequence encoding a Cry2Ab Bacillus thuringiensis δ-endotoxin protein, said second polynucleotide sequence being operably linked to a plant functional 3′ end transcription termination and polyadenylation sequence, wherein expression of said nucleic acid sequence in said plant yields a fusion protein comprising an amino-terminal plastid transit peptide covalently linked to said δ-endotoxin protein, wherein said plastid transit peptide functions to localize said δ-endotoxin protein to a plastid, and wherein said expression results in increased levels of said δ-endotoxin protein relative to plants transformed to express a nucleic acid sequence encoding a Cry2AB protein which is not localized to the plastid;
- b) obtaining a second plant;
- and
- c) crossing said first plant and said second plant to obtain a crossed transgenic progeny plant, said progeny plant having inherited said nucleic acid sequence from said first plant.
A nucleic acid sequence comprising in operable linkage a plant functional promoter linked to a first polynucleotide sequence encoding a plastid transit peptide and a second polynucleotide sequence encoding a Cry2Ab Bacillus thuringiensis δ-endotoxin protein, wherein expression of said nucleic acid sequence by a plant cell produces said δ-endotoxin protein localized within a plastid, and wherein said expression is increased relative to a plant cell transformed to express a nucleic acid sequence encoding a Cry2Ab protein which is not localized to a plastid.
A morphologically normal plant comprising a nucleic acid sequence comprising in operable linkage a plant functional promoter sequence linked to a first polynucleotide sequence encoding a plastid transit peptide and a second polynucleotide sequence encoding a Cry2Ab Bacillus thuringiensis δ-endotoxin protein, wherein said δ-endotoxin protein is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:18, and wherein expression of said nucleic acid sequence in said plant yields said protein localized to a plastid.
A morphologically normal plant comprising a nucleic acid sequence comprising, in 5′ to 3′ order, a plant functional promoter sequence operably linked to a first polynucleotide sequence encoding a plastid transit peptide, which is linked in frame to a second polynucleotide sequence encoding a Cry2Ab Bacillus thuringiensis δ-endotoxin protein selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:18, said second polynucleotide sequence being further linked to a plant functional 3′ end transcription termination and polyadenylation sequence, wherein expression of said nucleic acid sequence in said plant yields a fusion protein comprised of an amino terminal plastid transit peptide covalently linked to said δ-endotoxin protein, and wherein said plastid transit peptide functions to localize said δ-endotoxin protein to a plastid.
A plant cell comprising a nucleic acid sequence comprising a plant functional promoter sequence operably linked to a first polynucleotide sequence encoding a plastid transit peptide, which is linked in frame to a second polynucleotide sequence encoding a Cry2Ab Bacillus thuringiensis δ-endotoxin protein wherein said second polynucleotide sequence is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:18.
A seed produced from the plant of claim 56 or the progeny plant of claim 134, wherein said seed comprises said nucleic acid sequence.
Dependencies are inferred from the claim text.