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Fusion Expression and Biological Activity Analysis of cry1Ac Gene from Bacillus Thuringiensis with CDEP2 Gene from Beauveria Bassiana

Author: ZengZhi
Tutor: DingXueZhi;XiaLiQiu
School: Hunan Normal University
Course: Fermentation Engineering
Keywords: Bacillus thuringiensis Beauveria bassiana cry1Ac gene CDEP2 gene Red/ET homologous recombination Western blot
CLC: S476
Type: Master's thesis
Year: 2007
Downloads: 30
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Abstract


In order to obtain Bacillus thuringiensis（Bt）recombinant strain with higher toxicity and broader insecticidal activity spectrum,we construct the fusion gene of cry1Ac from Bt4.0718（CCTCC No.200016）with a new subtilisin-like serine protease gene CDEP2 from Beauveria bassiana to enhance the toxicity of Bt crystal proteins.Appling the way of Biology information to analyze the sequence homology of Beauveria bassiana subtilisin-like serine protease genes in GenBank,and RT-PCR amplify a new eDNA of subtilisin-like serine protease gene.We nominate this novel gene as CDEP2 for cuticle degrading enzyme proteinase2,the GenBank accession number is EF195164.It contains an ORF of 1137bp,encodes 379 amino acids with the predicted mass of 38.8KDa and isoelectrie point of 8.21.The ORF of CDEP2 gene was inserted into the fusion expression vector pET28a（+） and the recombinant plasmid was transformed into E.coli strain BL21. The（His）6-CDEP2 was expressed highly in BL21 induced by IPTG and purified by affinity chromatography.Immunization of rabbit with the CDEP2 protein generated high titer polyclonal antibodies.We take the pHT-cry1Ac-HwtxⅪas a target plasmid,then amplification CDEP2 that contained homologous arms.Using Red/ET homologous recombination,the Huwentoxin-Ⅺgene（HwtxⅪ）of the spider Selenocosmia huwena was replaced by CDEP2 gene in the recombinant plasmid.The recombinant vector pHT-cry1Ac-CDEP2 was electro-transformed into Bt acrystalliferous strain Cry- B.SDS-PAGE and Western blot analysis showed that fusion protein was expressed in the recombinant strain CryB-pHT-cry1Ac-CDEP2.It forms crystal more quickly and production more high.Bioassays analysis indicated that the CryB-pHT-cry1Ac-CDEP2 strain fermentation broth had high toxicity to the larvae of Helicoverpa armigera and the 50%lethal concentrations (LC50)after 72h was 8.50μl/ml,the control strain Cry- B-pHT-cry1Ac was 16.70μl/ml.This work successfully constructed the fusion gene contained cry1Ac gene and CDEP2 gene.It made a good ground for constructing the fusion genes of Bt cry gene and other foreign toxin genes.Red/ET homologous recombination technology takes a new idea for the foreign gene expression in Bt.

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CLC: > Agricultural Sciences > Plant Protection > A variety of control methods > Biological control
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