The Gene Cloning And Functional Analysis Of Rim15,Ume6 And Ime2 Gene In Nomuraea Rileyi

Nomuraea rileyi is an environment-friendly fungus that can infect various lepidoptera pests in the natural environment and is used as a new biological insecticide.However,the fermentation production of Nomuraea rileyi is very strict in terms of nutrition requirement,strict production cycle and high production cost,which limits the scale production and commercial application of Nomuraea rileyi.After long-term research,the success of the laboratory by liquid fermentation induced Nomuraea rileyi microsclerotium,because the microsclerotium has high production efficiency,lower production costs,product characteristics such as stable easy in stocking transportation,can continue to infect,pioneered the use of micro fungi sclerotia as a substitute for traditional conidium new ways of active ingredients.From the molecular level,the development mechanism of the microsclerotium of Nomuraea rileyi was studied,and it was of great theoretical and practical significance to understand the adaptability of fungi to the environment and to guide the growth of microsclerotium.Advance our laboratory on Nomuraea rileyi microsclerotium formation comparing the transcriptome sequencing found,in the process of microsclerotium forming Nomuraea rileyi Rim15,Ume6 and Ime2 are raised expression,Nomuraea rileyi spore germination in the process of microsclerotium forming,glucose concentration,hyphae polarity growth and oxygen stress can affect the generation of microsclerotium.Only some studies have found that Rim15 can integrate multiple pathways of nutritional signals,respond to glucose concentration,participate in spore formation,and respond to oxygen stress in different fungi.Ume6 was involved in hyphae polarity growth and infection.Ime2 was involved in spore formation,Pseudohypha growth and infection.However,the above mentioned genes were not reported in the Nomuraea rileyi,especially in the formation of microsclerotia.Accordingly,this research cloning NrRim15,NrUme6 and NrIme2 genes,and by building a knockout strains of mutant physiological morphology,cope with stress,microsclerotium formation and infect targets were analyzed,and the form is mainly to obtain the following results:(1)Gene cloning and sequence analysisThe full-length sequences of NrRim15,NrUme6 and NrIme2 were obtained from the genomic clones of Nomuraea rileyi,the login ID is OAA39692.1,OAA36838.1 and OAA44248.1,and the information analysis revealed that the open reading frame of NrRim15 was 5775 bp,encoding 1924 amino acids.The molecular weight of the protein was 210.63 KDa,the isoelectric point was 6.09,no signal peptide and transmembrane domain,containing fungal Rim15-like catalytic domain,PKc-like Superfamily protein kinase catalytic domain and REC signal receiving domain;NrUme6 open reading frame is 1839 bp,encoding 612 amino acids,the protein molecular weight is 67.35 KDa,the isoelectric point is 8.64,no signal peptide and transmembrane domain,contains GAL4-like protein binding region and Fungal_trans fungi specific transcription factor region;NrIme2 open reading frame is 2322 bp and encodes 773 amino acids,the molecular weight of the protein is 85.06 KDa,the isoelectric point is 9.55,no signal peptide and transmembrane domain,contains the catalytic domain of STKc_MAK_like protein kinase and the catalytic domain of PKc_like Superfamily protein kinase.Pairs and phylogenetic analysis revealed that the three genes were highly related to Metarhizium spp.(2)Expression patterns of Nomuraea rileyi at different developmental stages and under different conditionsThe qPCR analysis of the samples from two different transformation stages of Nomuraea rileyi on the SMSY plate revealed that the target genes were expressed in different degrees in the dimorphic transition of Nomuraea rileyi.NrRim15 and NrIme2 were highly expressed during the transition from the yeast state to the hyphae state,while NrUme6 was expressed at a lower level during the dimorphic transition process.During microsclerotia nucleus induction,NrRim15,NrUme6 and NrIme2 genes were expressed in all stages of microsclerotia formation.The expression levels of NrRim15,NrUme6 and NrIme2 were low in the early stage,and they all increased rapidly during the peak period of microsclerotia formation.Genes are involved in the formation of microsclerotia.10%,50%,and 90%(addition amount is the percentage of glucose in the conventional medium)glucose induction medium,liquid culture of Nomuraea rileyi microsclerotia to a large number of formation phase,qPCR quantification of microsclerotia samples The analysis found that the expression levels of NrRim15,NrUme6 and NrIme2 were not significantly different at different glucose concentrations.(3)Gene knockout vector construction and mutant strain screeningThe left and right flanks of NrRim15,NrUme6 and NrIme2 genes were amplified and the restriction sites were added.XhoI(single enzyme digestion)and XbaI(single enzyme digestion)were introduced into the left and right sides of NrRim15,Ecorl+XhoI(double enzyme digestion)and HindIII(single enzyme digestion)were flanked by NrUme6,and the left and right NrIme2 were introduced into EcoRI(single enzyme digestion)and XbaI.(Single enzyme digestion),analyzing the compatibility of restriction sites,selecting the best sequence for ligation,and successfully obtaining the knockout vector of the target gene.Agrobacterium tumefaciens genetic transformation system was used for co-cultivation to replace the target gene with the Hph fragment in the knockout vector.The stable inherited transformant strain was screened for Hph resistance,through the isolation of low concentrations of single spores,the target gene knockout mutants were successfully obtained: ?NrRim15,?NrUme6 and ?NrIme2.(4)Basic growth analysis of mutant strainsThe analysis of the culture characteristics of the three mutant strains revealed that the deletion of NrRim15 delayed the dimorphic transition of the strain,and the hyphae became thicker,longer and abnormally expanded,and the sporulation volume was significantly decreased.The loss of NrIme2 severely affected the growth rate of the strain.The hyphae growth was delayed,the cytoplasm was increased,the sporulation time was delayed,and the sporulation was slightly lower than that of the wild type.The deletion of NrUme6 was not significantly different from that of the wild-type strain.The spores of all mutants were not significantly different in morphology compared to the wild type,but the germination rate of conidia after NrRim15 deletion was significantly lower than that of the wild type,and the spore germination rate of NrUme6 and NrIme2 deletion strains was not significantly changed.(5)Mutant strains analyzed for the growth of different concentrations of glucoseIn different concentrations of glucose medium(the amount of glucose added in the conventional medium as a percentage),there was no significant difference in the size of the colony between the wild type and the mutant strains,but the NrRim15 mutant strain in the colony morphology was at a low concentration of 10% glucose carbon source Compared with the wild type,the transformation of the two types was significantly delayed,and the sporulation was 51.25% lower than that of the wild type.The higher concentrations(50% and 90%)of the colonies were similar to those of the wild type showing a white hyphae,indicating that NrRim15 was in the village of Nomuraea rileyi played a role in response to low concentrations of glucose,and NrIme2 and NrUme6 deletion strains did not differ significantly from the wild-type strains.(6)Mutant strains respond to stress growth analysisUnder ionic stress,addition of NaCl revealed that both NrRim15 and NrIme2 deletions resulted in strain growth limitation,and spore production was reduced by 88% and 94.67%,respectively,compared to basal medium;however,the growth and sporulation of NrRim15 and NrIme2 deletion strains were both increased after the addition of KCl.There were different degrees of recovery,in which spore production recovered 92.85% and 92.98%,respectively;NrUme6 deletion strain had no significant difference with wild type in ion stress.In osmotic stress,?NrRim15 was found to inhibit normal growth of glycerol and sorbitol stress,indicating that NrRim15 deletion resulted in increased susceptibility to osmotic stress;NrUme6 and NrIme2 deletion strains were more resistant to osmotic stress than anaerobic strains.No significant changes in type.The three mutant strains were insensitive to the cell disrupting agents SDS and CR.Only the addition of SDS to NrRim15 deletion was found to cause a small degree of inhibition of strain growth;the results of the oxygen stress assay showed that the medium after addition of hydrogen peroxide and menadione,the normal growth of ?NrRim15 and ?NrIme2 strains were significantly inhibited,and sporulation was 90.76% and 53.84% lower than that of the wild type strains.The growth of the two deletion strains after adding menadione was more inhibited.Strong,showing strong intolerance to oxidative stress;NrUme6-deleted strain showed no significant difference in growth and development with wild-type under oxygen stress,indicating that NrRim15 and NrIme2 genes play an important role in the oxygen metabolism of Nomuraea rileyi.The NrUme6 gene is not involved in this process.(7)Mutation Strain Induces Micro Sclerotia AnalysisThe fluid induced wild-type and mutant strains of microsclerotia.The results showed that the deletion of NrRim15,NrUme6 and NrIme2 had different effects on the normal formation of microsclerotia,showing that the formation time of microsclerotia was significantly delayed compared with the wild type,in which the diameter of nucleus of ?NrRim15 became smaller,compactness and pigmentation.The deposition decreased,and the mycelium on the edge of the microsclerotia was long and abundant.The yield and biomass of the microsclerotia decreased by 61.53% and 40.91%,respectively.The diameter of the nucleus of the ?NrUme6 decreased,the compactness decreased,and the yield and biomass of the microsclerotia decreased by 23.07.% and 9.09%;?NrIme2 micro sclerotia size was uneven,margin mycelium was short and sparse,micro sclerotia yield and biomass decreased by 10.05% and 18.18%,respectively.(8)Bioassay of mutant strainsThe third-instar larvae of Spodoptera litura were used as infective targets.The invasiveness of wild-type and mutants was determined by spot-injection and in vivo injection.The results showed that the mortality of the target larvae infected with NrRim15 and NrIme2 was lower than that of the wild type.In vivo injection experiments showed that the ?NrRim15 mutant and ?NrIme2 mutant strains had 5.6 and 2.4 days longer than the wild type,respectively.The surface infection assay showed that the NrRim15 and NrIme2 mutant strain had a longer lethal time than the wild type for 5.0 d and 3.5 d respectively.The survival rate of the larvae infected by the ?NrRim15 strain increased more significantly,and the half lethal time was longer.The half-lethal time of ?NrUme6 strain was similar to that of the wild type,showing a similar pathogenicity to the wild type.The Spodoptera litura larvae infected with the NrRim15 and NrIme2 mutants became significantly delayed in the process of becoming immobilised,suggesting that the loss of NrRim15 and NrIme2 resulted in a reduction in pathogenicity.There was no significant difference in the insects infected by ?NrUme6 strain compared with the wild type,indicating that the NrUme6 gene is not related to the virulence of Nomuraea rileyi to the host.Conclusion: The NrRim15,NrUme6 and NrIme2 genes play different roles in the growth and development of Nomuraea rileyi strains.Among them,NrRim15 and NrIme2 participate in the oxygen metabolism of the strains,and they have dimorphic transition,hyphae growth,sporulation and resistance to the strains.Both the power and the virulence of Spodoptera litura have different degrees of influence,while NrUme6 gene is almost not involved in the oxygen metabolism of the strain and has little effect on the two-type transformation,hyphae development,sporulation and virulence of the strain.All the three genes affected the development of microsclerotia.The knockout mutants decreased the yield and biomass of microsclerotia to varying degrees and affected the morphology of microsclerotia.However,its mechanism of action remains to be further studied.