CDNA Cloning And Prokaryotical Expression Of Carboxylesterase Genes In Locusta Migratoria

Locusta migratoria, belonging to Orthoptera Acridoidea, is a kind of worldwide agricultural pest which can be located in Asia, Oceania, Africa and Europe. Carboxylesterase is an important metabolic detoxification enzyme in insects and its main function is hydrolysis of endogenous and exogenous toxic substances containing ester bonds. In this thesis, we investigated CarE4, CarE9and CarE25in the levels of gene, molecular and protein, and demonstrated the characteristics of these three genes in different levels.In this thesis, we firstly searched the conserved region of the carboxylesterase gene in the ESTs database, then got the full length of CarE4after sequence finishing. On this basis, we designed the sense and antisense primers to amplifiy the full length of CarE4, and analyzed the information of CarE4in the levels of gene, primary and secondary structures of protein.After getting the full length of CarE4, we carried out the study of its mRNA expression by the method of real-time PCR. It showed that the expression of CarE4in every instar made a trend of firstly decreased and then increased, meanwhile, CarE4had a higher expression in gastric caeca compared with other tissues in L. migratoria.Then we carried out the research of prokaryotic expression of CarE4, CarE9and CarE25, and constructed three recombinant expression vectors (pCold TF-CarE4, pGEX-6p-1-CarE9and pET32M-CarE25), and revealed the best prokaryotic induced expression condition of these three carboxylesterase genes. On this basis, we purified these three proteins by three different methods and summarized the best approaches of purification of these proteins. In this thesis, we also prepared the antibody of CarE9protein, detected the expression of CarE9protein in9different tissues in L. migratoria by the method of Western Blot, which came to the conclusion that CarE9protein had a higher expression in gastric caeca than other tissues. In a word, this thesis researched CarE4, CarE9and CarE25in different levels, which not only made a certain reference value of detecting the characteristics of other carboxylesterase genes, but also provided the basic information for study the physiological function and structural characteristics of carboxylesterase in insect.