The Function Study Of Spore-related Genes VosA, VelB And Acy Of Arthrobotrys Oligospora

Arthrobotrys oligospora is a typical species of nematode-trapping fungi,which can finish reproduce asexually by producing conidium.At present,little is known about the mechanism of conidiation and regulatory genes for spore production in A.oligospora.In this study,three sporulation-related genes including VosA,VelB and Acy,were knocked out in A.oligospora,and their functions were preliminary analyzed.The main experimental results1.Analysis of the amino acid sequences and conserved domains of the three sporulation-related proteins in A.oligospora.The protein Acy has a relatively larger molecular weight and contains 2,100 amino acid residues(aa),while both of proteins VosA and VelB proteins have relatively small molecular weights,cantains 497 and335 aa,respectively.Both of VosA and VelB contain two conserved domains,including velvet factor(IPR011009)and velvet domain(IPR037525).Acy protein contains eight conserved domains,such as adenylyl cyclase class-3/4/guanylyl cyclase(IPR001054)and leucine-rich repeat domain superfamily(IPR032675).Cluster analysis showed that VosA,VelB and Acy in A.oligospora clustered with the homologus proteins in Aspergillus nidulans.2.Gene deletion of sporulation-related genes and analysis of phenotypic traits.Using the method of homologous recombination,three sporulation-related genes were successfully obtained,and the phenotypic characteristics of the wild type strain(WT)and the corresponding mutants were compared,including colony morphology,growth rate,formation and the number of traps,conidiation and activity of extracellular protease.The results showed that the growth rate of the ?Acy mutants was slower than the WT and the aerial mycelium was weaker than that of the WT.Meanwhile,the?Acy mutants could not produce conidia and traps,and the activity of extracellular protease was also reduced.Similarly,the growth rate of ?VelB mutants were distinctly slower than the WT,its colonies became more dense,and mycelia morphology changes obviously,the ?VelB mutants also could not produce conidia,and the nematocidal activity was significantly reduced.However,there were no significantchanges in phenotypes between the ?VosA mutants and the WT.3.Analysis of the transcriptional levels of several phenotypic traits reated genes in the ?VelB and ?Acy mutants.The mycelia of the WT,?VelB and ?Acy mutant strains were collected at 3,5 and 7 days,and the total RNAs were extracted and transcribed reversely into cDNA,respectively.Several genes related to conidiation,stress-resisting and extracellular protease were selected and their transcriptional levels were compared between the WT and mutants using RT-PCR method.In the ?VelB mutants,the transcription of most selected genes decreased singnificantly compared to the WT,suggested that the VelB gene can regulate the expression of these genes,which was consistent to the phenotypic differences.In ?Acy mutants,the transcriptional level of these tested genes also changed compared to the WT,which was consistent to the phenotypic differences.Interestingly,the trascription of these genes in the ?Acy mutants was down-regulated at 3 and 5 days,but their transcription restored at 7 day.These results indicate that VelB protein and Acy protein play important roles in these biological processes in A.oligospora.Innovations in this paper:1.The encoding genes of VosA,VelB and Acy were knocked out in A.oligospora,and the phenotypic traits of mutants were compared to the WT.Our results showed that VelB and Acy may involve in the regulation of important biological processes,such as growth,sporulation,and trap formation.2.The transcriptional levels of several genes related to conidiation,stress resistance and serine proteases were compared between the mutants(?VelB or ?Acy)and WT,and our results suggested that VelB and Acy may involve in the regulation of these genes expression in A.oligospora.