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AssigneeMonsanto Technology LLC
InventorsJames A. Baum, Amy Jelen Gilmer, Anne-Marie Light Mettus
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Disclosed are novel synthetically-modified B. thuringiensis nucleic acid segments encoding δ-endotoxins having insecticidal activity against lepidopteran insects. Also disclosed are synthetic crystal proteins encoded by these novel nucleic acid sequences. Methods of making and using these genes and proteins are disclosed as well as methods for the recombinant expression, and transformation of suitable host cells. Transformed host cells and transgenic plants expressing the modified endotoxin are also aspects of the invention. Also disclosed are methods for modifying, altering, and mutagenizing specific loop regions between the α helices in domain 1 of these crystal proteins, including Cry1C, to produce genetically-engineered recombinant cry* genes, and the proteins they encode which have improved insecticidal activity. In preferred embodiments, novel Cry1C* amino acid segments and the modified cry1C* nucleic acid sequences which encode them are disclosed.
A nucleic acid segment of from about 3567 to about 10,000 nucleotides in length, comprising a δ-endotoxin gene encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:59 or SEQ ID NO:61.
A composition comprising an isolated polypeptide that comprises the amino acid sequence of SEQ ID NO:59 or SEQ ID NO:61.
A composition comprising a polypeptide preparable by a process comprising the steps of:
A method of preparing a B. thuringiensis crystal protein comprising:
A purified antibody that specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:59 or SEQ ID NO:61.
A transgenic plant having incorporated into its genome a transgene that encodes a polypeptide comprising the amino sequence of SEQ ID NO:59 or SEQ ID NO:61.
A method of selecting a Cry1 polypeptide having increased insecticidal activity against a Lepidopteran insect comprising mutagenizing a population of polynucleotides to prepare a population of polypeptides encoded by said polynucleotides and testing said population of polypeptides and identifying a polypeptide having one or more modified amino acids in a loop region of domain 1 or in a loop region between domain 1 and domain 2, wherein said polypeptide has increased insecticidal activity against said insects.
A method of generating a Cry1 polypeptide having increased insecticidal activity against a Lepidopteran insect comprising the steps of:
A method of mutagenizing a Cry1 polypeptide to increase the insecticidal activity of said polypeptide against a Lepidopteran insect, said method comprising the steps of:
The method of claim 58, wherein said polypeptide is a Cry1C-R148D-K219A or Cry1C-R148A-K219Apolypeptide.
Dependencies are inferred from the claim text.