Functional Analysis Of Gα Gene In Nomuraea Rileyi

Nomuraea rileyi is an important entomopathogenic fungus.It can infect lepidopteran insects mainly in the Noctuidae by causing insect population epidemic to achieve control effect as a fungal insecticide.Gα protein encoded by Gα has been proved to be related to physiological activities,such as mycelium morphological differentiation,pathogenicity and synthesis of secondary metabolites.At present,there is no relevant report on the NrGα gene of Nomuraea rileyi at home and abroad.In this study,the Gα gene(NrGα)of Nomuraea rileyi CQNr01 strain was used as the research object.By constructing the Pzp-Ptrpc-Hph-Gα knockout vector,and using the Agrobacterium-mediated transformation technology,the ΔNrGα knockout strain was obtained.Conduct research on ΔNrGα growth and development,stress resistance,microsclerotia formation and insect pathogenicity to investigate the function of NrGαgene.The main research results are as follows:(1)NrGα of Nomuraea rileyi belongs to a class I Gα subunitsBioinformatics analysis shows that the open reading frame(ORF)of Gα is 1356 bp,encoding 370 amino acids,with 2 introns.The protein physicochemical properties predicted that the Gα gene encodes a 42.6KDa protein,does not contain transmembrane domains and is not a secreted protein.The results of homologous protein sequence alignment showed that the NrGα gene contains N-myristoylation site and ADP ribosylation site,which is consistent with the characteristics of class I Gα subunit and was identified as class I Gα subunit.(2)Knockout NrGα reduces the tolerance of Nomuraea rileyi to stress environmentThe stress resistance experiment found that ΔNrGα was sensitive to 100μg/ml CFw and 100μg/ml CR,and in addition to high concentration salt stress treatment,the mutant all showed normal sporulation,indicating that the NrGα gene plays an important role in maintaining cell wall integrity and sporulation.Microsclerotia is a dormant body structure by fungi to resist harsh environments.In the microsclerotia induction experiment,the number of ΔNrGα microsclerotia was reduced by 92.56% compared with the wild type,and the fluorescence quantitative results show that the pigment gene pks is low expressed,indicating that the NrGα gene inhibits microsclerotia formation,and affects the process of micronucleus nucleation and pigment deposition.(3)Knockout NrGα reduces the ability of Nomuraea rileyi utilize carbohydratesThe NrGα gene does not affect the intracellular c AMP content,but knockout NrGαreduces the ability of Nomuraea rileyi utilize carbohydrates,especially the use of glucose,fructose and trehalose.Indicating that knockout NrGα may affect the use of carbohydrates in c AMP-independent signaling pathway.(4)NrGα gene positively regulates the virulence of Nomuraea rileyiIn virulence experiments,the survival rates of ΔNrGα in vivo injection and surface infection were 92.22% and 98.89%,respectively.The reason may be that knockout NrGα makes the hyphal body use trehalose poorly,and the proliferation rate decreases,resulting in a decrease in the ability of ΔNrGα to escape insect immunity.(5)Transcriptome sequencing differential gene expression analysisTranscriptome sequencing during the dimorphic transition period of ΔNrGα,and a total of 27.07 Gb transcriptome sequences were obtained.Among the differentially expressed genes,1830 genes were up-regulated and 1794 genes were down-regulated.Among them,the expression of polar growth-related gene rac A was up-regulated,and the expression of cell wall gene Med A,cytoskeleton related protein(α-actinin and Dynein heavy chain),ph-response regulator protein pal A/RIM20,and cell division protein Cdc14 were all down-regulated.This indicates that there are several metabolic pathways involved in the process of transformation from yeast state to hyphae state.