| As an important entomopathogenic fungus,Metarhizium anisopliae plays a very important role in the biological control of various agricultural pests,and has a good development prospects.Current studies mainly focus on the surface infection of M.anisopliae,but relatively few on the digestive tract infection.As an important virulence factor,extracellular protease plays an important role in the infection of M.anisopliae.Previous studies found that the structure of microvilli in midgut of Locusta migratoria dropped off after feeding with M.anisopliae alone,while the structure of microvilli in midgut epidermis was intact after feeding with baits containing M.anisopliae and TPCK.In order to verify the destructive effect of extracellular protease in locust midgut and reveal the effect of extracellular protease on locust midgut immune response,the extracellular protease Pr1 C and Pr1 J of M.anisopliae were cloned and expressed.The effects of extracellular protease Pr1 C and Pr1 J on midgut immune response of locusts were verified by feeding with bait,which provided a theoretical basis for further revealing the mechanism of M.anisopliae.The main findings are as follows:1.Analysis of extracellular protease activity of M.anisopliae.After being treated with M.anisopliae and extracellular protease inhibitors,the midgut samples of L.migratoria were sequenced and analyzed by transcriptome sequencing.Differential gene analysis showed that 70 differentially expressed genes were screened between IPPM202 and IPPM202+TPCK based on P<0.05.22 genes were up-regulated and 48 genes were down-regulated.The up-regulated differentially expressed genes were mainly related to insect growth and development.The down-regulated genes were enriched in PI3K/Akt signaling pathway,which was related to insect innate immune response.It is concluded that extracellular protease plays a key role in the midgut immune response of the locust.2.Cloning and expression of extracellular protease gene and its biological activity assay.Two extracellular protease genes,Pr1 J and Pr1 C,were screened from the transcriptome of M.anisopliae.The sequence analysis of the two extracellular protease genes showed that the full length of the gene sequence was 1215 bp and 2124 bp,respectively.They encoded protein sequences containing 405 amino acids and 707 amino acids,respectively.They had high homology with subtilisin-like protease,reaching 99% and 97%,respectively.The similarity analysis showed that both of them contained the domain of Peptidase_S8 superfamily.Two kinds of extracellular proteases were obtained by isolation,cloning and prokaryotic expression.The bait feeding test showed that both proteases could enhance the virulence of M.anisopliae and reduce the survival rate of L.migratoria.3.Effect of extracellular protease on the immunity of L.migratoria.After feeding the bait containing extracellular protease and M.anisopliae,the changes of protease,detoxification and protective enzymes and immune-related gene expression in locust midgut were determined.The results showed that the activities of protective enzymes and detoxification enzymes of L.migratoria increased significantly when the extracellular protease Pr1 J or Pr1 C was mixed with M.anisopliae,however,the activity decreased gradually with the prolongation of treatment time.The expression of key genes of Toll and IMD pathway in the midgut of L.migratoria increased after the mixture of M.anisopliae and extracellular proteinase Pr1 J and Pr1 C.The expression of immune genes in the midgut of L.migratoria treated with Pr1 J,Pr1C and M.anisopliae was significantly higher than that of other treatments on the 2nd and 4th day,while the expression of genes in the midgut of L.migratoria treated with Pr1 J,Pr1C and M.anisopliae was significantly lower than that in the single place on the 6th day.Therefore,extracellular proteases Pr1 J and Pr1 C can induce locusts to increase their immunity to M.anisopliae,but the immunity gradually decreases with time. |
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