Cloning Of Chitin Synthase (CS) Gene From Ectropis Obliqua And Recombination Of Baculovirus With CS DsRNAi Gene

Ectropis obliqua Prout is a major pest in tea fields. Application of chemical pesticides in controlling the pests makes the pesticide residues in tea hang-up. Nucleopolyhedrovirus (NPV) is an effective biological pesticide to control the E. obliqua in tea field. However, short term and low efficacy limited the wide application of NPV in tea field. In this study, a full-length sequence of chitin synthase-1 (CS-1) cDNA from E. obliqua was cloned. A 192 bp conserved domain cloned from E. obliqua CS-1 gene was used to construct recombined Autographa californica M nucleopolyhedrovirus (AcMNPV) using double-stranded RNA interference (dsRNAi) method. The toxicity assessment of the recombinant showed that the CS-1 dsRNAi mediated by the NPV is peomising for development of alternative bio-pesticide to control the tea pest E. obliqua. Some other function genes were cloned for E. obliqua for further research.The cDNA sequence encoding the CS-1 and its expression pattern during development of E. obliqua were investigated. The CS-1 was expressed during the development of E. obliqua., and strong expressions were found in the third and fourth instar larvae, during which the growth rate of E. obliqua larvae was the rapidest. Catalysis model of CS enzyme in E. obliqua was also predicted according to the specific motifs and topological structure prediction of the protein. This study provided an important information for the further research on development of RNAi technology to control E. obliqua.The recombined AcMNPV was constructed with dsRNAi method using Bac-to-Bac system. The recombined AcMNPV virus could propagate in host cells sf9 and this can be used to develop an alternative method to control the E. obliqua in tea field. It is considered that the recombined AcMNPV virus control the E. obliqua by inhibiting the CS gene expression and chitin synthesis of the pest.Injecting and feeding tests using the recombined AcMNPV virus showed that the growth and development of both larvae and pupae of E. oblique were inhibited and the virus efficacy of the recombined AcMNPV on E. obliqua larvae was significantly higher than that of wild NPV. A dsCS2 recombined AcMNPV with the highest virus efficacy was screened from the tested viruses. The cumulative mortality of E. obliqua Ist instar larvae who were fed with the dsCS2 recombined AcMNPV virus was 95% on third day. It is considered that the dsRNAi mediated inhibition of the expression of gene CS in the pests. The study also showed that dsCS2 could infect other insects such as Bombyx mori and pomegranate-cankerworm. It shows that a foreign dsRNAi gene from one species can interference CS gene expression of a different species. The bio-pesticides developed by dsRNAi may have a broad-spectrum in pest-control.