Expression Analysis Of Esterase CsCaE026in The Field Population Of Chilo Suppressalis (Walker)(Lepidoptera:Crambidae)

The striped stem borer, Chilo suppressalis (Walker), is a major pest for rice production in China, which causes deadhearts and whiteheads and leads to yield loss. The esterase isozymes are relatively abundant in C. suppressalis and associated with the resistance to chemical insecticides. To explore esterase genes in C. suppressalis can provide the basis for monitoring and management of the resistance to insecticides in the fields, development of new efficient environmentally-friendly insecticides, and monitoring and control of pesticide residues in the environment. In this thesis, molecular cloning and expression analysis of an esterase gene, CsCaE026from C. suppressalis are conducted. The main results are summarized as follows:1. Molecular cloning and sequence analysis of C. suppressalis CsCaE026geneThe full-length sequence of the esterase gene, CsCaE026was cloned from C. suppressalis, which open reading frame (ORF) was1,671bp and coded557amino-acid residues, with a predicted molecular mass of63.66kDa and a calculated isoelectric point of5.86. A signal peptide of16amino acids, conserved domains of α/β-esterase family like Gly-x-Ser-x-Gly motif and the catalytic triad (Ser199, Glu323and His453), and two sites for disulfide bond formation (Cys82and Cys103) were predicted in the deduced protein sequence of CsCaE026. It was suggested that CsCaE026gene belongs to the subfamily of a-esterase.2. Prokaryotic expression of C. suppressalis CsCaE026geneThe Rosetta strain of Escherichia coli with recombinant plasmid of CsCaE026gene was induced and expressed as inclusion body. Its molecular weight was about70KD. However, the esterase activity of the recombinant protein has not been detected. The polyclonal antibody of the recombinant protein was prepared. The result of Western bloting confirmed that the CsCaE026gene was expressed successfully in E. coli.3. Temporal and spatial expression patterns of C. suppressalis CsCaE026gene The temporal and spatial expression patterns of CsCaE026gene at both transcriptional level and translational level in C. suppressalis were analyzed by real-time quantitative PCR and protein western bloting. The CsCaE026gene was expressed at high level in the midgut of C. suppressalis larvae.4. Eukaryotic expression of of C. suppressalis CsCaE026geneA recombinant baculovirus carrying the CsCaE026gene was constructed using Bac-to-Bac expression system and then used to transduce Sf9cells. The molecular weight of recombinant expression product was about90KD. The Western-bloting result showed that the gene was successfully expressed in Sf9cells.