Expression Of The Gryllus Bimaculatus ASTs Gene Concatemer In Bacillus Thuringiensis

ASTs protein is a member of insect Allatostatin neuropeptides family. It was isolated from the brain of Gryllus bimaculatus. In vitro, bioassay showed that ASTs protein greatly inhibited JH (Juvenile Hormone) biosynthesis by insect CA (corpora allata). The cDNA of ASTs precursor encodes fifteen ASTs. The nucleic acid sequences of each AST were named as AST1 gene, AST2 gene to AST15 gene by order, which are characterized by a common carboxy terminus (Y/FXFGL/I/V). This study was to construct a B.t- E.coli shuttle express vector and the ASTs was subcioned into B.t expression system to analyze its expression products. At the end, the expression products were applied to do an anti-insects experiment on Brontispa longissim.The cry3Aa gene and promoter fusing segment were amplified by PCR from the B.t-E.coli shuttle vector pHT305A with cry3Aa gene and its promoter, which were subcioned into the B.t- E.coli shuttle vector pHT304 in construction of the B.t- E.coli shuttle fusing expression vector pHT3 AP and pHT3AG.The AST1, AST2, AST5 and AST13 gene concatemer were linked to the multiple cloning sites of the B.t- E.coli shuttle expression vector pHT3AP and pHT3AG. After confirmed the reading frame of the recombinant plasmid by sequencing. Then the recombinant plasmid was transformed into B.t without plasmid mutant strain BMB171 for expression analysis. As a result, in the B.t without plasmid mutant strain BMB171, the target protein cry3Aa~ AST2 and cry3Aa—AST5 were respectively detected from the recombinant plasmid pHT3AG~ AST2 and pHT3AG~ AST5. SDS-PAGE analysis of the expression products indicated that the recombinant BMB171 strain expressed an additional protein with a molecular mass of 93—94kD, which was coincident with the molecular weight 94kD deduced theoretically.In the B.t expression system, the relation between cultivation time and expression quantities was studied for the recombinant plasmid pHT3AG—AST2 and pHT3AG— AST5; and the experiment results showed that the quantities of target protein was the highest for 60—72 hour cultivation time .The target protein cry3Aa~AST2 and cry3Aa~AST5 were used to do an...