Cloning, Expression And Functional Analysis Of Delta GSTs Gene In

The red flour beetle, Tribolium castaneum is not only a seriously harmful agricultural pest distributed all over the world, but also a representative insect for Coleoptera and a new model insect. Because of the insecticide resistance of Tribolium castaneum has been more and more serious, cause problems for food storage and food health. GSTs as one of the detoxification enzymes in organisms, most researches showed that GSTs is closely related to the insects resistant. Consequently, the research for relationship between GSTs and pesticide resistance of Tribolium castaneum has theoretical and practical significance, also play a foundation for the development of new pesticides. In this paper, we choosen three genes of Delta-class GSTs from Tribolium castaneum and start by sequence analysis, using real-time PCR, moleculer cloning、expression and protein purification technology、systemic RNA interference technology and susceptibility analysis for typical insecticide, made a preliminary analysis and inference for the function of TcGSTdl, TcGSTd2andTcGSTd3.The molecular cloning and successfully expressed in E.coli, sequencing analysis, laid the foundation for later research. We using real-time fluorescent quantitative PCR detected expression patterns in8developmental stages (early and late egg, larva, pupa and adult), the result shows TcGSTdl and TcGSTd2with significant expression specificity in developmental period. Both of them reached the highest in late pupa expression quantity compared with minimum expression period about8and40times respectively, and TcGSTd3gene reached the highest in late egg expression quantity compared with minimum expression period about3.6times.Larvae RNAi analysis showed that TcGSTdl, TcGSTd2and TcGSTd3gene silence resulting in total mortality was97%,4.8%and6.6%. Results also showed that the lack of TcGSTd1and TcGSTd3all lead to the reproduction dose was reduced by66%and55.1%respectively. And the knockdown of TcGSTd3causes the egg hatching rate of only1.8%, but TcGSTd1does not affect egg hatching, lack of TcGSTd2neither influence beetle’s reproductive also does not affect egg hatching.The total GSTs activity in larvae were dropped22.7%,15%and15.7%respectively after TcGSTdl, TcGSTd2and TcGSTd3were knockdown. And the resistance for phoxim and lambda-cyhalothrin were reduced in larval stage. The dsRNA treated larvae were exposed to phoxim (0.8%)48h, the mortality of experimental insect than the control group increased49%,37%and29%, respectively; and exposed to lambda-cyhalothrin (0.025%)72h, the mortality of experimental insect than the control group increased31%,22.5%and31%respectively. The result shows that TcGSTdl, TcGSTd2and TcGSTd3were able to metabolize or sequester lambda-cyhalothrin and phoxim in T.castaneum in different extent respectively.In addition, cloning and prokaryotic expression in vitro experiments show that recombinant proteins TcGSTdl, TcGSTd2and TcGSTd3can catalyze the conjugation of glutathione and CDNB, shows that the three recombinant proteins has the basic activity of glutathione S transferase, and participated in the insect detoxification.These results indicate that the all of TcGSTd1, TcGSTd2and TcGSTd3have basic activity of glutathione S-transferase, and participated in the insecticide resistance. Meanwhile, they also affected the beetle’s basic growth, development and reproduction. As TcGSTdl is a key gene for pupation, eclosion, growth and reproduction; and TcGSTd3may be involved in reproduction and embryonic development process. But TcGSTd2gene is mainly involved in detoxification of insecticides, it almost does not participate in the growth and reproduction in Tribolium castaneum.