Cloning Of Trypsin Upstream Promoter Core Sequence From The Midgut Of Mythimna Separata And Functional Verification Of It

With the focus on the food security and green agriculture,people are aware of safety use to pesticides.Therefore,the research and development of new pesticide with natural products has become one of the hottest spots in recent years.The preliminary study found that periplocoside compounds have certain insecticidal activity for Mythimna separata,which can up-regulate the expression of trypsin in vivo.The overexpression phenomenon of secretion exceeds the metabolic level of insect life itself,which may lead insects to death by making their own organs and tissues by the enzymatic hydrolysis of destruction.However,the present study has not yet established how compounds produce the effect,and the insecticidal mechanism has not been researched definitely.1.The deletion cloning of midgut trypsin gene promoter in Mythimna separataBioinformatics is used in the study to predict potential promoters and their binding site of midgut trypsin upstream gene promoter in Mythimna separata.According to the analysis results preliminary forecast results,the target promoter fragments were amplified by PCR with 7 pair of deletion primers,and recombinant plasmids were linearized into the luciferase reporter gene vector and they co-transfected to Sf21 insect cell lines.The relative fluorescence activity tested by transient expression showed the difference between activities of every promoter.The deletion cloning results showed that the target gene promoter region may contain certain transcription factor binding sites in-1003/-788 and-274/-188,which played a key role in trypsin gene,and there may be a transcription activators between promoter region-1003/-788,there may be a transcription repressor between promoter region-274/-188.2.The mutation cloning of midgut trypsin gene promoter in Mythimna separataAccording to the activity of deletion promoter and structure and function of transcription factors,6 potential transcription factors Zeste,Ubx,Myod,Cf2,SMAD,and STAT binding sites were predicted with bioinformatics method.These sequences of binding sites were mutated,and target promoter fragments were amplified by PCR with 6 pair of mutation primers.Recombinant plasmids that contained mutation promoter fragments were linearized into the luciferase reporter gene vector and co-transfected to Sf21 insect cell lines.The transient expression results showed that promoter activity was different from that in the blank control group after that binding sites of promoters Myod and Cf2.Therefore,the experiment showed that promoters Myod and Cf2 existed in vivo in Mythimna separata,and played an important role in the transcriptional regulation of midgut trypsin gene.3.The effect of periplocoside on target gene promoterIn order to explore whether periplocoside T compounds can have a direct effect on the target promoter,the tested drug was diluted to different concentration gradient by DMSO and acted to Sf21 insect cell lines that luciferase gene expression vector of whole promoter transfected to directly.After incubation for 5 hours,promoter activity was compared with blank control group.The results showed that the promoter activity of midgut trypsin gene in low concentration of periplocoside T decreased.With periplocoside T concentration increasing,promoter activity reduced.It showed that periplocoside T had an effect on promoter activity trypsin gene.Many methods were used to analyze and predict promoter of midgut trypsin gene in Mythimna separate in this study.With comparison of promoter activity between treatment group and control,potential transcription factor binding sites were found preliminarily.According to the interaction between periplocoside and target promoter,the results showed the compound had a certain effect on the target promoter,which laid a theoretical foundation for the mechanism of insecticides in the level of insect gene transcription and expression.