Neuropeptide Ast <sub> 13 </ Sub> Gene Series Body And Expression

ASTs protein is a member of insect Allatostatin neuropeptides family.It was isolated from the brain of Gryllus bimaculatus. In vitro, bioassay shows ASTs protein greatly inhibit JH (Juvenile Hormone) biosynthesis by insect CA (corpora allata). The cDNA of ASTs precursor encodes fifteen ASTs. The nucleic acid sequences of each AST are named as AST/ gene, AST2 gene to AST15 gene by order. The amino acid sequence of AST13 protein is N-TSDAFGL, adding up to seven residues. My major work was to construct an AST13 gene concatemer and then clone the concatemer into E. coli expression system to analyze its expression products. At the end, the expression products were applied to the beetarmyworm.Based on the encoding sequence of AST,3protein and the linking segment whose amino acid sequence is GKR, we synthesized two primers. One was named as L3, whose DNA sequence is 5' ACG TTT GCC CAG ACC GAA, and the other was named as A13, whose DNA sequence is 5' GGC AAA CGT ACC AGC GAT GCG TTC GGT CTG. Then they were used to construct a concatemer through a proved PCR method. In this procedure, high fidelity DNA Polymerase (Pyrobesf* DNA Polymerase) was used to ensure efficient and accurate amplification. After cloning and sequencing of the PCR products, one concatemer containing fifteen copies of ASTL1 gene was attained.The ASTin gene concatemer was coined into ?coli expression system, such as pET21lK pET22bK pET4^ pET30a^ IMPACT-en and pQESO, for expression analysis. But only pQE30 expression system, in which the expression level was high, met our request. We cloned the ASTU gene concatemer into pQESO expression vector. After confirmed the reading frame of the recombinant plasmid through sequencing, the recombinant plasmid was transformed into expression host SG10039 strain. Expression of AST13 gene concatemer was induced by IPTG. SDS-PAGE analysis of the expression products indicated that, compared with the control, the recombinant ?coli strain expressed an additional protein with a molecular mass of 19KD, which was coincident with the molecular weight 18. 8KD deduced theoretically. In expression analysis, we did some experiments on the concentration of IPTG and length of inducingtime for the highest expression level. The results of experiments showed that 0. 5mM/L was the best concentration and 1 hour was the best length of inducing time. Under the best conditions, the proportion during the aim protein and the total proteins was approximately 20. 2%. The expression products of AST]:) concatemer in pQESO expression system were used to do an anti-insects experiment on beet armyworm. At first, H2o, the expression products of pQE30 vector without foreign gene and the expression products of recombinant pQESO vector were mixed into the food for the beet armyworm respectively in the proportion of 1ml: lOg. Then, after the food was feeded to beet armyworm, we recorded the weight, length and death number of insects dayly from the third day. Bioassay showed the expression products of AST,:) gene concatemer have some toxicity to the beet armyworm.