The Gene Cloning And Functional Analysis Of NrPalI And NrPacC Gene In Nomuraea Rileyi

Nomuraea rileyi can infects a variety of lepidoptera pests under nature conditions,particularly the noctuid pests.It is an environmental friendly entomopathogenic fungus.Usually,the conidia of entomogenous fungi are the main effective components of Biopesticides.But special nutritional requirement and growth conditions are required for N.rileyi sporulation and its has long production cycle,which results in high costs that limits the large-scale production of N.rileyi.At the same time,its vulnerable to environmental factors(temperature,salt etc).Fortunately,N.rileyi has been successf?lly induced to produce environmentally persistent microsclerotia(MS).Due to its low cost,shelf-stable,resistance to adverse environments and good prevention effect,MS can replace conidia in practical application.Thus,the mechanism of MS development and elucidating the molecular mechanism of multi-stress tolerance contribute to its further application in biological control.Subsequently,Our findings show pH is change regularly in microenvironment during microsclerotia development by compare the transcriptome technology.Meanwhile,the gene of NrPacC and NrPalI associated with pH regulation are found in the transcriptome library of the two type conversion.Thus,the study of NrPacC and NrPalI gene function can provide theoretical guidance for the liquid fermentation of microsclerotia.So far,there is no relevant research report in N.rileyi.In this study,we clone the gene of Nr PacC and NrPalI from CQNr01 according to comparative transcriptomic library.The function of NrPac C and NrPalI gene was studied by single gene knockout via Agrobacterium tumefaciens-mediated genetic transformation in N.rileyi.The results were as follows:(1)Cloning and Sequence Analysis the PalI and PacC genes in N.rileyiAccording to the Unigene sequence,which is functionally annotated in transcriptome library,the two genes were first amplified from the CQNr01.They were named NrPacC and NrPalI.The accession numbers were OAA38885.1 and OAA42720.1,respectively.Sequence analysis showed that the open reading frame(ORF)of NrpacC gene was 2028 bp,which encode 592 amino acids and contain two introns(168bp,81bp).The open reading frame of NrPalI gene was 2103 bp,that encodes 576 amino acids and contain one intron with a length of 62 bp.Bioinformatics analysis indicate that the NrPacC gene encodes a 64.433 KDa protein with an isoelectric point of 8.66,which contain three Cys2His2 zinc finger structures that specifically bind to the 5'-GCCARG sequence of the target gene promoter region.The NrPalI gene encodes 61.398 KDa protein with an isoelectric point of 9.23,that has three transmembrane domains and a conserved domain of the SUR7 family.BLAST searches and Phylogenetic analysis demonstrated that NrPacC and NrPalI acid sequence share 23.4%~91% identity to homologous protein from other fungi and that NrPacC and NrPalI highly homologous to those of Metarhizium Robertsii and Metarhizium guizhouense,respectively.(2)Construction of NrPacC and NrPalI gene knockout vectors and screening of mutant strainsThe respective flanking regions of NrPacC and NrPalI were amplified through chromosome walking by the method of FPNI-PCR and the resulting upstream/downstream flanking sequences were finally inserted to knockout vector,yielding Pzp-Ptrpc-Hph-NrPacC and Pzp-Ptrpc-Hph-NrPalI respectively.The mutant strains ?NrPacC and ?NrPalI were obtained by Agrobacterium tumefaciens mediated transformation of N.rileyi.(3)Phenotypic analysis of mutant strains of NrPacC and NrPalIThe deletion of NrPacC and NrPalI genes has no effect on the morphology of conidia but leads to abnormal mycelial morphology.The knockout mutant strain of the two genes significantly delayed the transformation from the yeast state to the mycelial state and reduced the yield of conidia on the minimal medium.Under alkaline conditions,the growth of NrPacC knockout strain was inhibited obviously and the sporulation rate was decreased.However,NrPalI mutant strain was not significantly different.The results showed that NrPalI gene deletion was not sensitive to oxygen stress and metal ion stress.But,under the condition of cell wall perturbing agents,the initial colony growth was inhibited,then,there was no significant difference between relative to wild type.Meanwhile,the yield of sporulation descended significantly under osmotic stress and salt stress.The sporulation of NrPacC mutant strain decreased significantly in different stress conditions and the two types of conversion time were prolonged under H2O2 and fluorescence White stress.Under the stress of sodium and lithium,the growth of NrPacC mutant was obvious inhibited and sporulation decreased significantly.During the development of MS,the gene-deficient strains of NrPacC and NrPalI produced a large number of mycelium in the early stage and deepened the pigmentation of the MS.The mycelium of NrPalI mutant strain was twine earlier to form microsclerotia and the yield of MS increase dramatically.The microsclerotia of NrPacC mutant strain is larger than that of wild type.But there was no significant difference in biomass and microsclerotia yield contrast to wild type.(4)Expression patterns analysis of NrPacC and NrPalI genesThe expression levels of NrPacC and NrPalI genes at different pH conditions and the development of microsclerotia were performed by RT-qPCR.NrPacC and NrPalI genes were up-regulated expression trend from acidic(pH = 5),neutral(pH = 7)to alkaline(pH = 9)conditions.The expression levels of NrPacC and NrPalI were enhanced by 8 and 6.5 fold under alkaline conditions than acidic conditions,respectively.These data indicated that NrPacC and NrPalI genes were involved in the regulation of the alkaline environment.NrPacC and NrPalI genes were expressed in all stages of microsclerotium formation,but NrPacC and NrPalI transcribed dramatically increased at 4 d,which was up-regulated 12 fold than that of 2.5 d.Thus,these results indicated that NrPacC and NrPalI genes may be involved in microsclerotium development,including the stage of microsclerotium initiation and formation.(5)Toxicity analysis of ? NrPacC and ? NrPalI strainsThe virulence of the mutants was tested by inoculation onto the cuticle and injection in vivo of third-instar Spodoptera litura larvae.The results showed that the half lethal time(LT50)of NrPacC-deficient strains was 9 ± 0.6 d and 9.5 ± 0.8 d,which was about 2 d longer than that of wild type,while NrPalI mutant had no significant difference with wild type.Observation of muscardine cadaver by conidia infection found that the muscardine cadaver surface were covered mycelial by wild type injection,while ? NrPacC and ? NrPalI mutant strains no hypha growth in 7 days.At the time of 10 days,the muscardine cadaver have produced abundant conidia by wild type strain and NrPalI mutant strain infection,but the dead Spodoptera litura larvae by the NrPacC mutant strain invasion only part of the mycelium penetrate from the cutic?Lar layer and has no conidia.The results indicated that the deletion of NrPacC gene resulted in decreased virulence,while NrPalI knockout strain had no significant effect on the mortality of Spodoptera litura larvae.