| Spodoptera litura is a pest of the genus Spodoptera(Lepidoptera:Lepidoptera).It has the characteristics of short life cycle,overlapping generations,rapid reproduction,large fecundity,easy outbreak,miscellaneous food habits and strong drug resistance,so it is the main pest harmful to global agriculture.Entomopathogenic fungi are an important part of entomopathogenic microorganisms and play an important role in biological control.Metarhizium rileyi has become a biocontrol strain with wide application prospect and great development potential because of its advantages such as environmental friendliness,active infection of host,cyclic infection of spores and difficulty of host resistance.However,at present,there are few studies on the pathogenic mechanism of M.rileyi which limits its application in the biological control of pests such as S.litura.In this study,the research group previously identified the protein secreted by M.rileyi into the blood cavity of the host,and OAA36685 was selected as the research object to analyze its function in parasitic S.litura infected by M.rileyi.The specific experimental results are as follows:1.Gene cloning and sequence analysisThe nucleic acid fragment of the target gene OAA36685 was cloned and sequenced with M.cDNA as the template.The physical and chemical analysis of the gene showed that it had a complete open reading frame(ORF)of 1896 bp,encoding 631 amino acids,and the molecular weight of the protein was 71.003 kDa.There is a signal peptide(1-17 aa),but no transmembrane domain.It contains a peptidase superfamily M60 domain,which is inferred to be a member of the metallothionein family.Sequence alignment showed that the similarity of channel-related proteins between OAA36685 and Metarhizium anisopliae was the highest,which was 40.28%.2.Construction of knockout and complement vectors and screening to obtain mutationsThe OAA36685 gene knockout vector was constructed,and the Agrobacterium tumefaciens containing OAA36685 knockout vector was co-cultured with wild-type strains by Linear cassette knockout method,and the ΔOAA36685 knockout mutants were screened.Then the OAA36685 gene complementation vector was constructed.Transferring the complement vectors into Agrobacterium tumefaciens,and after co-culture with ΔOAA36685 knockout mutants,ΔOAA36685cp complementary strains were screened.3.Effect of knockout of OAA36685 Gene on the growth and Development of M.rileyiThe spore germination rate,growth rate and spore production of wild-type strain,ΔOAA36685 mutant and ΔOAA36685cp complement strain of M.rileyi were determined.The results showed that there was no difference in growth and development betweenΔOAA36685 mutant and ΔOAA36685cp complement strain and wild-type strain,indicating that knockout of OAA36685 gene did not affect the growth and development of M.rileyi,and the gene is not involved in the process of growth and development.4.Effect of knockout of OAA36685 gene on virulence of fungiThe third instar larvae of S.litura were naturally infected and the sixth instar larvae of S.litura were infected by injection with wild type strain,ΔOAA36685 mutant andΔOAA36685cp complement strain respectively.The death number of larvae was counted and the half death time was calculated.The results showed that the death time of larvae naturally infected with ΔOAA36685 mutant was longer,that is,the death rate was slower,and the semi-lethal time was 14 hours longer than that of wild type group.The results of injection infection showed that the death rate of larvae infected with ΔOAA36685 mutant was significantly lower than that of wild type group and supplement group,and its semi-lethal time was 4 hours longer than that of wild type group.These results suggested that the virulence of M.rileyi decreased after knockout OAA36685 gene,which was related to the virulence of M.rileyi.5.The effect of knockout of OAA36685 gene on the ability of M.rileyi to inhibit the spreading of plasmatocytes of host insects.The sixth instar larvae of S.litura were infected with conidia of wild type,ΔOAA36685 mutant or ΔOAA36685cp complementary strain,and the length of plasmatocytes was measured.The results showed that the length of plasmatocytes of the larvae infected with ΔOAA36685 mutant was significantly longer than that of wild type group and complementation strain group.The results suggested that the ability of M.rileyi to inhibit the plasma cell extension of S.litura decreased after the knockout of OAA36685 gene,and the OAA36685 gene played an important role in inhibiting the cellular immunity of host insects.6.The effect of knockout of OAA36685 gene on the ability of fungi to inhibit the activation of prophenoloxidase in host insects.The sixth instar larvae of S.litura were infected with conidia of wild type,ΔOAA36685 mutant and ΔOAA36685cp complementary strain.The serum of the larvae was taken to determine the activity of phenoloxidase at four stages after infection.The results showed that the phenoloxidase activity of the larvae infected with ΔOAA36685 mutant was significantly lower than that of wild type group and complementation strain group.The results suggested that M.rileyi inhibited the activation of prophenoloxidase in 5.litura after knockout OAA36685 gene,and OAA36685 gene played an important role in inhibiting humoral immunity of host insects.7.The effect of knockout OAA36685 gene on the number of hyphal bodies in host hemolymph after fungal infection.The sixth instar larvae of S.litura were infected with conidia of wild type,ΔOAA36685 mutant and ΔOAA36685cp complement strain.The serum of the larvae was collected at three stages after infection.The results showed that the number of hyphal bodies of the larvae infected with ΔOAA36685 mutant was significantly lower than that of wild type group and complementation strain group.The results suggested that the growth and reproduction of M.rileyi was inhibited in S.litura larvae after knockout OAA36685 gene,which may be caused by the decrease of immune inhibition ability of M.rileyi to host insects after knockout OAA36685 gene.To sum up,this study confirmed that OAA36685 has immunosuppressive activity and can inhibit the cellular immunity and prophenoloxidase activation response of hostinsects.These results lay a foundation for further study on the pathogenic mechanism of M.rileyi infecting host insects in the future,and provide potential target genes for obtaining more virulent strains of M.rileyi through genetic improvement in the future.It also has application value in using M.rileyi to control pests such as S.litura. |
PDF Full Text Request