Prokaryotic Expression,Tissue Localization And Binding Ability To Nonvolatile Compounds Of Chemosensory Protein CSP2 Of Spodoptera Litura

Spodoptera litura is a global agricultural pest,which is difficult to predict and control due to its migratory,polygou,multi-generation and nocturnal characteristics.Its offspring reproduction is exponential growth and cause great harm.In recent years,the extensive use of chemical pesticides has strengthened the resistance of Spodoptera litura to common chemical pesticides,and the pesticide "3R" has become more and more serious.Therefore,the use and development of new botanical pesticides is a matter of great urgency.The research of the sensing mechanism of Spodoptera litura to botanical pesticides is an important link in the development of new botanical pesticides.Chemosensory proteins are an integral part of the whole insect’s sensory system.Research shows that chemoceptor proteins play an important role in the action mechanism of non-volatile plant pesticides against Spodoptera litura.In this study,we studied the expression,localization and binding properties of CSP2 in larvae and adults of Spodoptera litura.The main conclusions are as follows:1.The cloned SlCSP2 was used to construct p ET-32a(+)vector and transferred into BL21(DE3)competent bacteria.After centrifugation,the supernatant was discarded in the induced Escherichia coli solution,and then the bacterial lysate was added for ultrasonic crushing.After centrifugation,the inclusion body was purified by affinity chromatography on the SlCSP2 recombinant protein with nickel column,and after dialysis with dialysate,the soluble SlCSP2 recombinant protein with biological activity was finally obtained.2.The purified SlCSP2 protein was used as antigen,and the polyclonal antibodies were immunized into New Zealand white rabbits to obtain the antibodies.The titer and specificity of the antibodies were detected with SlCSP2 antibody serum and His labeled primary antibody by Western blot.In order to investigate the tissue expression characteristics of SlCSP2,the m RNA expression levels of SlCSP2 in larval inages,pupal stages and adult tissues were detected by fluorescence quantitative PCR,and the protein expression levels of SlCSP2 in larval inages and adult tissues were detected by Western blot.The results showed that SlCSP2 was expressed in the six instars of larvae,pupal stage and adult tissues,and the expression of SlCSP2 showed a certain pattern in both larval and adult stages.At larval stage,the expression level of the third instar larvae was the highest and gradually decreased from the third instar to the sixth instar.In the adult stage,the expression levels of both females and males were high in the leg,thorax and head,but low in the antennae,and the expression levels in the tissues of males were generally higher than those of females.3.The expression and distribution of SlCSP2 protein in the antennae and leg of adult and six instars of Spodoptera litura larvae were analyzed by Western blot and immunofluorescence localization.The results showed that at larval stage,SlCSP2 protein was mainly distributed around the head,the mouth organ and the toe finger of the foreleg,and there was not much expression on the body hair.In the adult stage,SlCSP2 protein was mainly distributed in the spinous and conical sensilla of the antennae,but not in the trichoid sensilla,and the overall expression of SlCSP2 protein was not obvious in the antennae.In the leg,SlCSP2 was widely distributed in the front tarsal,especially in the toe hook,and was also expressed in other parts,which was consistent with the results of fluorescence quantitative PCR.4.In order to investigate the binding properties of SlCSP2 protein,the fluorescence competitive binding method was used to determine the binding properties of SlCSP2 protein with 13 common non-volatile compounds.The results showed that SlCSP2 protein had strong binding ability with tannin,anthocyanin and folic acid.In this study,SlCSP2 recombinant protein was expressed and purified,and the corresponding polyclonal antibody serum was prepared,and the tissue localization and binding properties of SlCSP2 protein were studied,which provided data support for biochemical control of Spodoptera striata.