Chimeric cry1E δendotoxin and methods of controlling insects
View Patent ↗A chimeric Cry1E δ endotoxin and methods of using it to treat plants to reduce or control insects.
1. A chimeric nucleic acid comprising SEQ ID NO:2.
2. The chimeric nucleic acid as claimed in claim 1 , wherein said chimera encodes a chimeric protein of SEQ ID NO:1.
3. The chimeric nucleic acid as claimed in claim 1 , wherein said chimera has a of length 1990 base pairs (bp).
4. The chimeric nucleic acid as claimed in claim 1 , wherein said chimera is a 1.99-kb double stranded DNA.
5. The chimeric nucleic acid as claimed in claim 1 , wherein said chimeric nucleic acid has ATGGCT at its 5′ extreme, as an initiation codon.
6. The chimeric nucleic acid as claimed in claim 1 , wherein said chimeric nucleic acid has TAATGA as a translation termination codon.
7. The chimeric nucleic acid as claimed in claim 1 , wherein said chimera has a T m value ranging between 44 to 55° C.
8. A method of overexpressing an insecticidal chimeric Cry1E protein in a microbe, wherein said method comprises:
(a) cloning SEQ ID NO:2, which encodes the protein, in a vector such that SEQ ID NO:2 is operably linked to a promoter,
(b) transforming a microbe with said vector, and
(c) overexpressing said chimeric Cry1E protein in said microbe.
9. The method as claimed in claim 8 , wherein said chimera is expressed into a microbe selected from the group consisting of bacteria, algae, and fungi.
10. The method as claimed in claim 8 , wherein the restriction enzymes for said cloning are selected from the group comprising of Hind III, EcoR1, Ncol, Mfe I and BamHI.
11. The method as claimed in claim 8 , wherein the promoter is inducible by isopropylthiogalactoside (IPTG) and wherein the protein is overexpressed by induction by IPTG.
12. The method as claimed in claim 8 , wherein the protein is overexpressed at 15° C. to avoid mis-folding of said protein.
13. The method as claimed in claim 8 , wherein said vectors are selected from the group consisting of plasmids, viral DNA, and cosmids.
14. The method as claimed in claim 8 , wherein expression of said protein in the microbe is confirmed by RT-PCR, Western analysis, and ELISA.
15. The method as claimed in claim 8 , wherein presence of SEQ ID NO:2 in the microbe is confirmed by PCR and Southern analysis.
16. A method of inducing insect tolerance into a plant wherein said method comprises:
incorporating a chimeric gene comprising the nucleic acid of claim 8 operably linked to a plant promoter into a plant, thereby producing a transgenic plant with insect tolerance.
17. The method as claimed in claim 16 , wherein said plant is selected from the group consisting of tobacco, cotton, chickpea, pigeonpea groundnut, cauliflower, cabbage, and capsicum.
18. The method as claimed in claim 16 , wherein restriction enzymes for said cloning are selected from the group consisting of Hind III, EcoRI, Ncol, and BamHI.
19. The method as claimed in claim 16 , wherein the chimeric protein is expressed at a level of 0.5% of the total soluble protein in the plant.