Screening Of Esterase Genes Related To Detoxification Metabolism In Chilo Suppressalis(Walker) And Their Expression

The rice stem borer, Chilo suppressalis (Walker), is one of major insect pests on rice in China. Especially in southern China, this pest outbreaks year by year and always causes huge economic losses. Generally, rice stem borer was controlled mainly by chemicals. Organophosphate and carbamate insecticides were widely used in practice. However, long-term extensive use of insecticides, especially unreasonable use, provided well selection pressure which made rice stem borer developed different levels of resistance to commonly used insecticides in China.Pest resistance mechanism involves penetration ability decrease, detoxification capacity enhancement and target insensitivity. Mutation, amplification and increased expression of esterase genes are all important mechanisms for detoxification capacity enhancement in insects. Esterase is encoded by a huge gene super-family, which is not only involved in detoxification of exogenous compounds, but also nutrition metabolism, signal transduction, Juvenile hormone metabolic regulation and some other important functions. With the transcriptome data, esterase gene fragments of C. suppressalis were screened and verified by PCR. Then, insecticide-induced experiments were carried out to select the genes which were possibly involved in the metabolism of special insecticide. Finally, the full-length sequences of the insecticide inducible esterase genes were cloned. The results obtained were summarized as follows which should find the basis for further analysis of the roles of special esterase in metabolism of various pesticides.1. Screening and verification of the esterase genes in C. suppressalis and analysis for those insecticide-inducibleIn order to identify the esterase involved in the detoxification of a special insecticide in C. suppressalis, the transcriptome data were used to screen for esterase sequences, and RT-PCR was used to verify them. As results,68different carboxyl-esterase gene fragments were obtained. Then, insecticide-inducing experiments with triazophos and chlorantraniliprole were performed, and the induction of all the68esterase genes were checked by using Semi-quantitative RT-PCR. According to the brightness of electrophoresis bands, the insecticide inducible esterase genes in C. suppressalis were identified. The results showed that among the68esterase genes, Cs.EXE2,Cs.EXE4,Cs.EXE5and Cs.EXE6could be induced by chlorantraniliprole, and Cs.EXEJ,Cs.EXE3,CsEXE4,Cs.EXE5could be induced by triazophos. Analysis came to the conclusion that these genes might be involved in the detoxification of these two insecticides, and encode the esterase functioning in insecticide detoxification metabolism.2. Cloning of the full length esterase genes insecticide-inducible in C. suppressalis and their sequence analysisFull-length sequences of the6esterase genes insecticide-inducible were cloned from C. suppressalis with Rapid Amplification of cDNA Ends procedure. They are all about1700bp-2500bp in length, with an open reading frame (ORF) about1400bp-1700bp, encoding about479-560amino acid. Except for Cs.EXE2, all Cs.EXEl,Cs.EXE3,Cs.EXE-4,Cs.EXE5and Cs.EXE6contains catalytic triads Ser-Glu-His, and catalytic center GXSXG. Sequence alignment showed that these genes all shears about84%-99%similarity with those reported esterase genes of Bombyx mori, Drosophila melanogaster. Apis mellifera and Tribolium castaneum. These results further confirmed that these6genes cloned are esterase genes of C. suppressalis.3. The expression analysis about detoxification esterase geneThere are a lot of esterase genes in C. suppressalis, and all share high sequence similarity. In order to avoid the possible errors in transcriptome fragment screening and semi-quantitative RT-PCR test, the cloned full length of esterase genes and real-time PCR were used to confirm the selected six insecticide-inducible esterase genes. The experiments got a similar result as above. Chlorantraniliprole induction made the expression level of the4insecticide-inducible esterase genes selected by semi-quantitative RT-PCR test increased by2.5-3.5times. Triazophos induction made them increased by1.5-7.5times. These results further confirmed that these six gens were insecticide-inducible in C. suppressalis. Discussion came to the deduction that these six insecticide-inducible esterase genes might be involved in the detoxification metabolism of chlorantraniliprole and triazophos.