The Gene Cloning And Functional Analysis Of Nrssk1 And Nrpbs2 In Nomuraea Rileyi

Nomuraea rileyi(Metarhizium rileyi)is a widely distributed entomopathogenic fungus.Because of its ability to infect a variety of Lepidoptera pests in natural environment,it gets a growing attention as it can be used as a potential bio-pesticide.However,the sporulation conditions of the conidia are very harsh and are not conducive to storage,which is an important factor restricting the industrial production of fungi with conidia as an active ingredient of insecticidal preparations.Our laboratory has successfully realized the artificial liquid induction of Microsclerotia(MS).The production cost of MS is low,and it is of high temperature resistance,storage resistance and sustainable infection,so it can be used as an effective substitute for conidia for the production of insecticidal preparations.Our laboratory used to study the the molecular mechanism of the MS formation by comparative transcriptome technology,and we found that MS develops along with the form of reactive oxygen species(ROS).There are researches show that the two transmembrane proteins,Sholp and Sln1 p,play an important role in the response of the fungi to external osmotic stress and oxygen stress.The two proteins transmit the signal to the high osmolarity glycerol(HOG).On the other hand,previous studies have shown that the genes of sho1 and sln1 of Nomuraea rileyi play an important role in responding to osmotic pressure and oxygen stress and regulating microflora development.Given this,Nrssk1 and Nrpbs2 in HOG pathway was successfully cloned according to the transcriptome information;and the two genes were successfully knocked out by agrobacterium-mediated transformation,so we can study genic functions in this paper,and the main results show as follows:(1)Gene cloning and sequence analysis of Nrssk1&Nrpbs2The partial sequences of Nrssk1 and Nrpbs2 genes were cloned successfully according to the Unigene sequences of Nomuraea rileyi.Then the full length of the genes were amplified by FPNI-PCR.We found that the ORF of Nrssk1 is 2929 bp with 2 introns,encoding 929 amino acids,the protein molecular weight is 100.2kDa and the theoretical isoelectric point(PI)is 10.04.The ORF of Nrpbs2 is 2038 bp,containing an intron,encoding 649 amino acids,the protein molecular weight is 68.5kDa and the theoretical PI of the simulated protein is 5.80.The secondary structure of the genes is dominated by random coil and ? helix.Nrssk1 p belongs to the REC superfamily,which contains the signal receiving domain;Nrpbs2p belongs to the PKc_like superfamily,which is composed mainly of serine / threonine specificity and catalytic domain of tyrosine-specific protein kinase.By multiple sequences alignment and phylogeny evolution analysis,we found that Nrssk1 and Nrpbs2 are most closely related to Metarhizium in evolutionary relationship by multiple sequences alignment and phylogeny evolution analysis.(2)Single knockout of Nrssk1&Nrpbs2The flanking sequences of Nrssk1&Nrpbs2 were amplified by FPNI-PCR,and about 1500 bp sequences which are unknown were obtained respectively,so we can construct Pzp-Ptrpc-Hph-Nrssk1 and Pzp-Ptrpc-Hph-Nrpbs2.The mutants ?Nrssk1 and ?Nrpbs2 were selected by ATMT techniques.(3)Phenotypic characterization of mutantsThe results showed that the growth of ?Nrpbs2,especially the pre-conidial germination,was later than that of the wild type,the mycelium was richer and the conidiophore was more,and it got a trouble with conidiation while the quantity decreased sharply.?Nrssk1 didn't have a significant change compared to wild type.In the stress experiment,?Nrpbs2 was sensitive to salt stress,mycelial growth was inhibited,and the limit tolerance to salt concentration was decreased.In addition,?Nrpbs2 was more sensitive to osmotic stress,and the morphologies of the colony changed obviously.Its conidia are smaller than wild type.The growth of ?Nrpbs2 was restricted by oxygen stress,especially in the oxygen stress environment provided by menadione,and the growth process was obviously delayed.And the colonies of ?Nrssk1 got an obvious change compared with wild type only under a high degree of osmotic stress and oxygen stress,the growth of the strain was limited,the amount of sporulation decreased in varying degrees.In addition,both mutants were insensitive to damp heat stress.In MS induction experiment,the MS pigmentation of ?Nrssk1 was decreased,and the compactness of MS became looser.The changes above got more obviously in ?Nrpbs2,we can perceive the pigmentation change under the macro observation.Moreover,the two mutant strains had a significant decrease in MS yield(quantity)and biomass.The results above show that the two genes are necessary in response to various stresses and the development of MS.(4)Expression patterns analysis of Nrssk1&Nrpbs2 during MS developmentThe samples were collected at several stages of MS development and the expression levels of the two genes were analyzed by real-time PCR,the results showed that the two genes had similar expression trends.At the early 36 th hour of the induction of MS,both genes had higher expression levels and then decreased,and reached the highest expression level at 84 th hour.At this time,it was at the peak of MS formation,which indicated that the two genes are involved in the development of MS and play a certain role.Induce MS with mutants to investigate their synergistic relationship by real-time PCR,and we found that the two genes were synergistic to some extent and complemented by CWI pathway.(5)Virulence detectionThe conidial spores of the mutants were infected with the third instar larvae of the genus Spodoptera littoralis by inoculation onto the cuticle and in vivo injection.The results showed that the half-lethal time of ?Nrssk1 was 6.18 d and 6.43 d,respectively,1.18 d and 1.36 d longer compared with the wild type.And the LT50 of was 6.83 d and 6.97 d respectively,1.83 d and 1.90 d longer compared with the wild type.This indicated that both genes affected the virulence to the Spodoptera litura larvae to some extent.(6)ConclusionThe results above indicate that these two genes play a role in dealing with the stress,development of MS and virulence,and the function of the two genes is similar,but the effect is not the same,which is consistent with the position of the two genes in HOG pathway.