| Serine protease inhibitors(Serpins)are the most widely distributed and the largest class of inhibitor superfamily,which play an important role in the immune response of insects to pathogens mainly by regulating the activity of proteases.The serpins family has long been shown to play a major role in humoral immunity.Locusta migratoria manilensis is one of the most important pests in the world,which causes serious harm to agriculture and animal husbandry.Previous laboratory studies showed that Metarhizium anisopliae had a good control effect on locust migratory fungus,but the immune mechanism of locust migratory was less studied.L.migratoria manilensis in this paper,in order to study the serine protease inhibitors in the immune mechanism in the resistance of M.anisopliae bacteria,cloning and acquire Serpin1 protein,were examined by using the method of feeding protein and RNAi bassiana fungus after processing the mortality and the body parts of L.migratoria manilensis protective enzyme and detoxifying enzyme activity,and the determination of the fat in the body immune related genes expression character,in order to further reveal the immune mechanism of L.migratoria manilensis mission is based.The results are as follows:1.The expression level of serpin1 gene was detected by q RT-PCR,which indicated that serpin1 was involved in the immunity of L.migratoria manilensis.serpin1 was expressed in all the instars and tissues of the L.migratoria manilensis,and the highest expression was found in the third instar and integument.Serpin1 purified protein was obtained by cloning and prokaryotic expression. Sequence analysis of the Serpin1 gene showed that the sequence of the serpin1 gene was 1228bp and the open reading frame was 1040 bp,encoding 346 amino acids and the molecular weight of the protein was 35.6k Da.2.The inhibitory effect of serpin1 protein on the enzyme system and protease in M.anisopliae was verified by plate culture and in vitro and in vivo enzyme activity tests.The results showed that Serpin1 protein at different concentrations could not inhibit the growth of M.anisopliae,but could significantly inhibit the activity of extracellular protease in M.anisopliae.In vitro validation test showed that different contents of Serpin1 protein could significantly inhibit the activities of trypsin and chymotrypsin,and 10μg of Serpin1 protein had the highest inhibitory effect.3.Through L.migratoria manilensis Serpin1 protein and M.anisopliae fungus mixed treatment, validation of M.anisopliae fungus insecticidal effect,and is verified by enzyme standard method between different treatment groups protective enzyme(SOD,POD),detoxification enzymes(GSTs, MFO),hydrolase(ESTs,ACh E),and the activity of the PO,q RT-PCR verification Toll pathways related gene(toll,myd88,tube,sp?tzle,persephone,defensin)and melanization of the expression of related gene(PPAE,PPO)research.The results showed that the mixed treatment of Serpin1 with M.anisopliae significantly reduced the insecticidal effect of M.anisopliae against locusts.Serpin1 protein and M.anisopliae could significantly increase the activities of protective enzyme,detoxification enzyme,hydrolase and PO,and the expression of genes related to Toll pathway and melanization reaction in the L.migratoria manilensis.4.Again after the serpin1 by interfering with the insect body,applying M.anisopliae,and is verified by enzyme standard method between different treatment groups protective enzyme,detoxification enzymes,hydrolase,and the activity of the PO,q RT-PCR verification Toll pathways related genes and mechanism related genesresearch.Studies have shown that interference with serpin1 can increase the mortality of L.migratoria manilensis.The results showed that the activities of 7 enzymes in vivo were disregulated,and the expressions of 7 related genes in vivo of L.migratoria manilensis were decreased and increased.In other words,serpin1 can enhance the enzymatic immunity of L.migratoria manilensis and improve the expression of immune-related genes. |
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