Plants transformed with polynucleotides encoding broad-spectrum delta-endotoxins
Disclosed are novel synthetically-modified B. thuringiensis chimeric crystal proteins having improved insecticidal activity and broader insect host range against coleopteran, dipteran and lepidopteran insects. Also disclosed are the nucleic acid segments encoding these novel peptides. 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.
1. A transgenic plant having incorporated into its genome a polynucleotide encoding an insecticidal Bacillus thuringiensis hybrid crystal protein comprising domains I and II of a first native B. thuringiensis delta-endotoxin Cry1A protein, domain III of a second native B. thuringiensis delta-endotoxin Cry1F protein, and all or a portion of a protoxin segment of a native delta endotoxin Cry1A, Cry1F or combination Cry1F-Cry1A protein.
2. The transgenic plant of claim 1 , wherein said polynucleotide hybridizes under high stringency conditions of about 0.02 M NaCl to about 0.15 M NaCl at about 50° C. to about 70° C. with a sequence which is or is complementary to the sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:33.
3. A progeny of the transgenic plant of claim 1 , wherein said progeny has incorporated into its genome a polynucleotide encoding an insecticidal Bacillus thuringiensis hybrid crystal protein comprising domains I and II of a first native B. thuringiensis delta-endotoxin Cry1A protein, domain III of a second native B. thuringiensis delta-endotoxin Cry1F protein, and all or a portion of a protoxin segment of a native delta endotoxin Cry1A, Cry1F or combination Cry1F-Cry I A protein.
4. A seed from the transgenic plant of claim 1 , wherein said seed has incorporated into its genome a polynucleotide encoding an insecticidal Bacillus thuringiensis hybrid crystal protein comprising domains I and II of a first native B. thuringiensis delta-endotoxin CryIA protein, domain III of a second native B. thuringiensis delta-endotoxin Cry1F protein, and all or a portion of a protoxin segment of a native delta endotoxin Cry1A, Cry1F or combination Cry1F-Cry1A protein.
5. A seed from the progeny of claim 3 , wherein said seed has incorporated into its genome a polynucleotide encoding an insecticidal Bacillus thuringiensis hybrid crystal protein comprising domains I and II of a first native B. thuringiensis delta-endotoxin Cry1A protein, domain III of a second native B. thuringiensis delta-endotoxin Cry1F protein, and all or a portion of a protoxin segment of a native delta endotoxin Cry1A, Cry1F or combination Cry1F-Cry1A protein.
6. A plant from the seed of claim 5 .
7. The transgenic plant of claim 1 , wherein said insecticidal Bacillus thuringiensis hybrid crystal protein comprises an amino acid sequence at least 90% identical to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:34.
8. The transgenic plant of claim 1 , wherein said insecticidal Bacillus thuringiensis hybrid crystal protein exhibits increased insecticidal activity against an insect, relative to either of the non-hybrid proteins from which it was engineered; and the insect is a member of an insect family selected from the group consisting of Heliothis, Helicoverpa, Pectinophora, Spodoptera , and Earias.
9. The transgenic plant of claim 8 , wherein said insect is a species selected from the group consisting of Heliothis virescens, Helicoverpa zea, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua, Spodoptera frugiperda, Earias vitella , and Spodoptera litura.
10. The transgenic plant of claim 1 , wherein said insecticidal Bacillus thuringiensis hybrid crystal protein is isolatable from a Bacillus thuringiensis cell selected from the group consisting of NRRL B-21579, NRRL B-21580, NRRL B-21581, NRRL B-21635, NRRL B-21636, and NRRL B-21781.
11. The transgenic plant of claim 1 , wherein said transgenic plant is corn, wheat, oat, rice, maize, barley, cotton, soybean, ornamental, shrub, fruit, nut, vegetable, turf grass, pasture grass, berry, or citrus.
12. A transgenic plant having incorporated into its genome more than one transgene encoding more than one insecticidal Bacillus thuringiensis hybrid crystal protein comprising domains I and II of a first native B. thuringiensis delta-endotoxin Cry1A protein, domain III of a second native B. thuringiensis delta-endotoxin Cry1F or Cry1C protein, and all or a portion of a protoxin segment of a native delta endotoxin Cry1A, Cry1F, Cry1C, or combination Cry1F-Cry1A protein.
13. The transgenic plant of claim 12 , wherein said more than one transgene include two or more of cry1Ac-IF, cry1Ab-IF, cry1Ac-1C, and cry1Ab-1Ac-1F.
14. The transgenic plant of claim 12 , wherein said more than one insecticidal Bacillus thuringiensis hybrid crystal protein each comprises a different amino acid sequence which is at least 90% identical to SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34.
15. The transgenic plant of claim 12 , wherein said more than one transgene each hybridizes under high stringency conditions of about 0.02 M NaCl to about 0.15 M NaCl at about 50° C. to about 70° C. with a sequence which is or is complementary to the sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 and SEQ ID NO:33.
16. The transgenic plant of claim 12 , wherein said transgenic plant exhibits increased insecticidal activity against a broad spectrum of insects, relative to a non-transgenic plant comprising either of the non-hybrid proteins from which said transgenic plant was engineered; and said broad spectrum of insects are members of one or more insect families of Heliothis, Helicoverpa, Pectinophora, Spodoptera and Earias.
17. The transgenic plant of claim 16 , wherein said broad spectrum of insects are one or more species of Heliothis virescens, Helicoverpa zea, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua, Spodoptera frugiperda, Earias vitella , and Spodoptera litura.
18. The transgenic plant of claim 12 , wherein said more than one insecticidal Bacillus thuringiensis hybrid crystal protein each is isolatable from a Bacillus thuringiensis cell selected from the group consisting of NRRL B-2 1579, NRRL B-21580, NRRL B-21581, NRRL B-21635, NRRL B-21636, NRRL B-21780 and NRRL B-21781.
19. The transgenic plant of claim 12 , wherein said transgenic plant is corn, wheat, oat, rice, maize, barley, cotton, soybean, ornamental, shrub, fruit, nut, vegetable, turf grass, pasture grass, berry, or citrus.
20. A progeny of the transgenic plant of claim 12 , wherein said progeny has incorporated into its genome more than one transgene encoding more than one insecticidal Bacillus thuringiensis hybrid crystal protein comprising domains I and II of a first native B. thuringiensis delta-endotoxin Cry1A protein, domain III of a second native B. thuringiensis delta-endotoxin Cry1F or Cry1C protein, and all or a portion of a protoxin segment of a native delta endotoxin Cry1A, Cry1F, Cry1C, or combination Cry1F-Cry1A protein.
21. A seed from the transgenic plant of claim 12 , wherein said seed has incorporated into its genome more than one transgene encoding more than one insecticidal Bacillus thuringiensis hybrid crystal protein comprising domains I and II of a first native B. thuringiensis delta-endotoxin Cry1A protein, domain III of a second native B. thuringiensis delta-endotoxin Cry1F or Cry1C protein, and all or a portion of a protoxin segment of a native delta endotoxin Cry1A, Cry1F, Cry1C, or combination Cry1F-Cry1A protein.
22. A seed from the progeny of claim 20 , wherein said seed has incorporated into its genome more than one transgene encoding more than one insecticidal Bacillus thuringiensis hybrid crystal protein comprising domains I and II of a first native B. thuringiensis delta-endotoxin Cry1A protein, domain III of a second native B. thuringiensis delta-endotoxin Cry1F or Cry1C protein, and all or a portion of a protoxin segment of a native delta endotoxin Cry1A, Cry1F, Cry1C, or combination Cry1F-Cry1A protein.
23. A plant from the seed of claim 22 .