Immune Regulation Mechanism Of LmserpinB3 Gene On Locusts Migratory Against Metarhizium Anisopliae Infection And Its DsRNA Application

Serine protease inhibitors(serpins)are a superfamily of inhibitors that regulate the immune system of insects,mainly by modulating the activity of various serine proteases in the Toll signalling pathway and the melanization pathway to regulate insect antimicrobial peptides and the clearance of pathogenic bacteria.The Locusta migratoria is the migratory worldwide pest that causes serious damage to agriculture and animal husbandry.Studies have shown that Metarhizium anisopliae has a control effect on L.migratoria,but there are few studies on the resistance mechanism of L.migratoria to M.anisopliae,as well as the mechanism of serpins in L.migratoria immunity.In order to investigate the mechanism of serpins in the immune response of L.migratoria,the transcriptome of L.migratoria was analysed,and all the serpins in L.migratoria were screened and analysed,A representative gene(Lmserpin B3)was selected to investigate the biological function.RNAi technology and M.anisopliae infection were used to explore.And the changes of gene expression,protective enzyme and detoxification enzyme activities in toll pathway and melanization had been determined when Lmserpin B3 was interfered with alone and co-treated with M.anisopliae.Also the application potential of Lmserpin B3 as a control target was evaluated by measuring the effect of interference with Lmserpin B3 on the efficiency of M.anisopliae to control L.migratoria.The results are as follows:1)Through the screening of the transcriptome of L.migratoria,a total of 18 serpins genes were found,all of which had only one serpin domain and high gene conservatism.Subcellular localization analysis showed that except for Lmserpin B8-1,which was only localized in the cytoplasm,the rest were localized in both the cytoplasm and extracellular positions.After 24 h and48h infection with M.anisopliae,except Lmserpin B10 and Lmserpin B6-2,the expression of other genes had different changes.Through species evolution analysis,it was found that the gene conservatism of the Serpin family in the same species was high,and the conservatism between species was not high.2)The Lmserpin B3 gene was cloned and induced,and the results showed that the Lmserpin B3 gene sequence was 1221 bp,encoding a total of 406 amino acids and a protein molecular weight of 48.6 KDa.It was expressed in all instars and tissues of L.migratoria,and the highest expression was in the first instar and epidermis.3)After Lmserpin B3 interfered alone,and co-treatments,the changes in the expression of Toll signaling pathway regulatory genes(GNBP,Toll,Myd88,Sp?tzle,Tube,Relish,Defensin)and melanization related genes(PPO,PPAE)and the protective enzymes(SOD,POD,CAT),detoxification enzymes(GSTs,MFO)activity and survival rate of L.migratoria after different treatments were determined,.The results showed that Lmserpin B3 interference and M.anisopliae infection mainly reduced the expression of insect antimicrobial peptides(Defensin),PPO,PPAE and the activity of detoxification enzymes.Co-treatments,Lmserpin B3 interference and M.anisopliae infection,reduced the survival rate of L.migratoria compared with M.anisopliae infected alone.4)The dsRNA expression vector of Lmserpin B3 gene was constructed to obtain dsRNA expressed by Escherichia coli(E.coli).By means of bait-feeding dsRNA,the effect of gene interference was exerted to improve the control effect of M.anisopliae on L.migratoria by about30%.Therefore,Lmserpin B3 can be used as a biological control target for L.migratoria,and its dsRNA can be co-administered with M.anisopliae to accelerate the effective rate and improve the lethality of L.migratoria.