The Analysis Of Transcriptional Difference To Isaria Fumosorosea Stimulation And Prokaryotic Expression Of Serpin-14 From Plutella Xylostella

Plutella xylostella,as a major agricultural pest and mainly harms Cruciferae vegetables,in the past years approximately US$4 billion annually were used in his management.Due to excessive and unwise use of synthetic pesticides,not the only environment is disturbed but also strong ability of resistance has been developed in P.xylostella against all classes of synthetic insecticides,therefore the new field of interest should be focused.Biological control is now being very significant method having no environmental and resistance issues.Serine protease inhibitors can regulate various physiological process via mediate the corresponding Protease and can inhibit the excessive melanization also.The current study was planned to further explore the function of Serpin in P.xylostella.The main results are as follows:1.In order to better investigate the immune system of P.xylostella,the forth,instar P.xylostella were treated and the samples were collected after 12 h,18h,24 h,and 36 h.These samples from the data of transcriptomes were used for RNA-Sequencing.We found 21 Serpin with differential expression.In the four time periods of 12 h,18h,24 h,and 36 h,8,9,14,7 genes were up-regulated respectively and 12,12,7,14 genes were down-regulated respectively.2.The relative expression of Serpin-14 in Hemocyte,Fat body,Epidermis,and Midgut was detected by real-time quantitative PCR.The results showed that Serpin-14 is almost exclusively expressed in Hemocyte,which positively showed that most Serpin reacted in the hemolymph.At the same time,the expression level of the Serpin-14 in the whole development stages from egg to adult was also detected.The results showed that the expression level of Serpin-14 in the third and fourth instars was higher than other instars,it is probably related to its own development.3.In order to clarify the structure of the Serpin-14 protein,the protein tertiary structure was constructed by using some software,and the RCL located,in its C-terminal active region was identified,which offer a good reference to further research its target protein.its signal peptide cleavage site was predicted and located in 19 th amino acid.By using the NJ method,a phylogenetic tree was build and the results showed that the Serpin-14 from diamondback moth were high homologous to Serpin-14 from Operophtera brumata.4.In this study for further investigation of the function of Serpin-14 from protein level in P.xylostella,we successfully expressed the prokaryotic expression vector of pET32a-Serpin-14 in BL21(DE3).The relative molecular mass of pET32a-Serpin-14 protein was detected by SDS-PAGE and Western blot techniques.The pET32a-Serpin-14 protein was purified by Ni-NTA column chromatography,and then purified protein was immunized into rabbits to prepare polyclonal antibodies,which laid a foundation for subsequent studies on protein levels.P.xylostella was treated with Isaria fumosorosea and the transcriptomes were sequenced at different time points after treatment in this study.The relative expression of different development stages and different tissues of Serpin-14 were detected by real-time quantitative PCR.At the same time,we have analyzed the amino acid sequence of Serpin-14,and polyclonal antibody was prepared.