Nucleic acid molecules encoding insecticidal proteins

Patent metadata

Field	Value
Publication number	US-10167323-B2
Application number	US-201514963898-A
Country	US
Kind code	B2
Filing date	Dec 9, 2015
Priority date	Oct 2, 2009
Publication date	Jan 1, 2019
Grant date	Jan 1, 2019

Abstract

Improved compositions and methods for controlling pests are disclosed. In particular, nucleic acid molecules encoding novel engineered Cry1Ba (eCry1Ba) proteins having improved toxicity to lepidopteran insect pests are provided. By substituting at least one amino acid in domain I of a Cry1Ba protein an engineered Cry1Ba protein having substantially altered insecticidal properties is designed. Further, methods of making and using the ecry1Ba nucleic acid sequences, for example in transgenic plants to express eCry1B proteins to confer protection from insect damage are disclosed.

First claim

The invention claimed is:

1. A nucleic acid molecule comprising a nucleotide sequence that encodes an engineered Cry1Ba (eCry1Ba) insecticidal protein comprising a mutation at a position corresponding to amino acids 2 and 150; or amino acids 2, 150 and 178; or amino acids 2, 150 and 189; or amino acids 2, 150 and 199, of SEQ ID NO: 5, wherein the amino acid corresponding to position 2 is any amino acid and (a) the amino acid corresponding to position 150 is Lys; or (b) the amino acid corresponding to position 150 is Lys and the amino acid corresponding to position 178 is Ser; or (c) the amino acid corresponding to position 150 is Lys and the amino acid corresponding to position 189 is Ser; or (d) the amino acid corresponding to position 150 is Lys and the amino acid corresponding to position 199 is Lys, and wherein said protein has improved solubility or insecticidal activity against at least European corn borer ( Ostrinia nubilalis ) when compared to a native or wild-type Cry1Ba protein, and wherein optionally the nucleotide sequence is optimized for expression in a transgenic host cell.

2. The nucleic acid molecule of claim 1 , wherein the protein comprises an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

3. The nucleic acid molecule of claim 1 , wherein the eCry1Ba protein has activity against lepidopteran or coleopteran insects.

4. The nucleic acid molecule of claim 3 , wherein the lepidopteran insects are selected from the group consisting of European corn borer, southwestern corn borer, sugarcane borer, corn earworm, soybean looper and velvet bean caterpillar.

5. The nucleic acid molecule of claim 1 , wherein the transgenic host cell is a bacterial cell or a plant cell.

6. The nucleic acid molecule of claim 5 , wherein the plant cell is a maize plant cell.

7. The nucleic acid molecule of claim 6 , wherein the nucleotide sequence comprises SEQ ID NO:13.

8. A chimeric gene comprising a heterologous promoter sequence operatively linked to the nucleic acid molecule of any one of claim 1 or 2 - 7 .

9. The chimeric gene of claim 8 , wherein the promoter is a plant-expressible promoter.

10. The chimeric gene of claim 9 , wherein the plant-expressible promoter is selected from the group consisting of ubiquitin, cmp, corn TrpA, mtl, bacteriophage T3 gene 9 5′ UTR, corn sucrose synthetase 1, corn alcohol dehydrogenase 1, corn light harvesting complex, corn heat shock protein, pea small subunit RuBP carboxylase, Ti plasmid opine synthase, Ti plaid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, Potato patatin, lectin, CaMV 35S, and the S-E9 small subunit RuBP carboxylase promoter.

11. A recombinant vector comprising the chimeric gene of claim 10 .

12. The vector of claim 11 , wherein the vector is a plasmid, cosmid, phagemid, artificial chromosome, phage or viral vector.

13. A transgenic non-human host cell comprising the chimeric gene of claim 8 .

14. The transgenic host cell of claim 13 that is a bacterial cell or a plant cell.

15. The transgenic host call of claim 14 , wherein the bacterial cell is an E. coli, Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium; Bacillus cereus, Agrobacterium or a Pseudomonas cell.

16. The transgenic host cell of claim 14 , wherein the plant cell is a corn, wheat, oat rice, barley, sugar cane, turf grass, pasture grass, cotton, sunflower, cole crop vegetable, sugar beet, tobacco, or an oilseed rape cell.

17. A transgenic plant comprising the plant cell of claim 16 .

18. The transgenic plant of claim 17 , wherein the plant is a monocotyledonous plant or a dicotyledonous plant.

19. The transgenic plant of claim 18 , wherein the monocotyledonous plant is selected from the group consisting of corn, wheat, oat, rice, barley, sugar cane, turf grass and a pasture grass.

20. The transgenic plant of claim 19 , wherein said plant is a corn plant.

21. The transgenic plant of claim 18 , wherein the dicotyledonous plant is selected from the group consisting of soybean, cotton, sunflower, cole crop vegetable, sugar beet, tobacco, and oilseed rape.

22. A progeny of any generation of the plant of claim 17 , wherein the progeny comprises the nucleic acid molecule.

23. A seed from any generation of the transgenic plant of claim 17 , wherein the seed comprises the nucleic acid molecule.

24. A method of making an insect-resistant transgenic plant, comprising introducing the nucleic acid molecule of claim 1 into a plant thereby producing a transgenic plant, wherein the nucleic acid molecule causes the expression of the engineered Cry1Ba protein in an amount that renders the transgenic plant insect resistant.

25. The method of claim 24 , wherein the introducing step is carried out by transforming a plant with the nucleic acid molecule.

26. The method of claim 24 , wherein the introducing step is carried out by crossing a transgenic plant comprising the nucleic acid molecule with a different plant.

27. The method of any one of claims 24 - 26 , wherein the plant is maize.

28. The method of claim 24 , wherein the plant is resistant to lepidopteran insects.

29. The method of claim 28 , wherein the lepidopteran insects are selected from the group consisting of European corn borer, southwestern corn borer, sugarcane borer, corn earworm, soybean looper and velvet bean caterpillar.

Assignees

• Syngenta Participations Ag

Inventors

• Gao Yan
• Conville Jared
• Chen Jeng Shong

Classifications

• C07K14/325 Primary

  Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins · CPC title

• A01N47/44

  Guanidine; Derivatives thereof · CPC title

• C12N15/8286 Primary

  for insect resistance · CPC title

• A01H5/00

  Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy · CPC title

• Y02A40/162

  Cross-Sectional Technologies · mapped topic