The Studies On Truncated Proteins Of Cry1Ie1 From Bacillus Thuringiensis

Plutella xylostella and Oatrimia fitrnacalis are important pests in agriculture. The resistance of P. xylostella to chemical pesticides has been rapidly increasing in recent years. O.furnacalis is the key stem borer in corn. The conventional strategies for controlling them was not effective. The protein expressed by cryllel, a silent gene from Bacillus thnringiensis in China, showed high toxicity to P. xylostella and O.jurnacalis. We constructed seven deleted Cryllel porteins and determined its minimum toxic regionAccording to the full sequence of cryllel gene, seven pair of primers( P001/P656. P001/P659,P001/P648, P001/P634, P045/P656, P105/P656,P045/P648) were designed to amplify different DNA fragment of cryllelgene. Seven deletions with different length 1968bp, 1977bp, 1944bp, 1902bp, 1833bp, 1653bp, 1812bp were obtained respectively. These DNA fragments were inserted into expression vector pET-21b and expressed in E.coli BL21(DE3). Seven truncated proteins were obtained, and they are IE656(lacking 63 amino acids of the C-terminal), IE659(lacking 60 amino acids of the C-terminal), IE648(lacking 71 amino acids of the C-terminal ), IE634(lacking 85 amino acids of the C-terminal ), IE045(lacking 44 amino acids of the N-terminal and lacking 63 amino acids of the C-terminal), lE105(lacking 104 amino acids of the N-terminal and lacking 63 amino acids of the C-terminal), IE045-648(lacking 44 amino acids of the N-terminal and lacking 71 amino acids of the C-terminal).The bioassay of these seven truncated proteins was conducted. The results indicated that the toxicity of IE656, IE659 to O. furnacalis was similar to that of the full length Crvllel protein. The toxicity of IE648 increased by 60% while that of IE045 decreased by 93.75%. IE 105 and IE634 did not show toxicity to O.jurnacalis. These results suggested that the minimum toxic region to O.jurnacalis was located between 634 and 648 amino acid of C-terminal, and 44 amino acids of the N-terminal were necessary for the activity.The toxicity of IE656, IE659 to P. xylostella was slightly lower than that of full length Cryllel protein, while IE648 and IE045 showed 30% higher toxicity than it. At the same time IE105 and IE634 was not toxic to P. xylostella. These results revealed that the minimum toxic region to P. xylostella was located between 45 and 104 amino acid of N-terminal, and between 634 and 648 amino acid of N-terminalCandidate: Wu Yu e Major: Botany Supervisor: Prof. Gao Ji guo...