Construction And Biological Characteristic Research Of The Asf1 Gene Knockout Mutant In Stemphylium Eturmiunum

Stemphylium eturmiunum is one of the important groups of filamentous fungi,which is one of the few Stemphylium species capable of sexual reproduction.It has mating type genes MAT1-1 and MAT1-2.That provides a good material for studying the sexual reproduction mechanism and evolution of fungi.asf1(anti-silencing function 1)is an evolutionarily highly conserved heterodimer histone H3/H4 chaperone.asf1 is involved in the processes of assembly and disassembly of nucleosomes that rely on DNA replication and are dependent of DNA replication.It is also involved in the processes of gene silencing,transcriptional regulation and DNA damage repair.asf1 has functional conservatism and diversities in different organisms.Histone chaperones regulate the expression of genes and affect the development of organisms by mediating the structure,stability and chromosome state of nucleosomes.However,few researches have been done on the asf1 gene in filamentous fungi.Therefore we study the S.eturmiunum asf1 and explore its role in the development of this fungi.In this study,Stemphylium eturmiunum(EGS 29-099)was selected as the experimental material,which contains two mating genes MAT1-1 and MAT1-2.S.eturmiunum can be induced to reproduce sexually in the CM medium under the 25 ?conditions.The developmental characteristics of it are the production of black perithecia and ascospores.In order to study the effects of asf1 gene on the S.eturmiunum development,asf1 gene was knocked out.The transcriptomes of S.eturmiunum wild type strains and asf1 gene knockout transformants were sequenced and analyzed for trying to explore the connection between phenotypes and intrinsic molecular mechanism.The main results are as follows:The S.eturmiunum asf1 gene was successfully cloned and identified.Then the asf1 gene sequence was analyzed by bioinformatics.Constructing the knockout vector of asf1 gene.And asf1 gene knockout transformants were obtained.Next,the biological characteristics of asf1 gene knockout transformants were studied.The control was wild type strains.It was found that the knockout transformants were asexual.?asf1 grown very slow and their hyphae had many branches.The phenomenon of ?asf1 hyphal fusion was very common.The conidia shape of ?asf1 changed from conventional spherical or oval into irregular shape.The conidia of ?asf1 were pruduced from on the hyphae to on the hyphae tipcease and they even changed into chlamydospore.The number of conidial septum was less.The conidia were most fasciclation and single microspore became larger.The conidiophores of ?asf1 were developed by mycelia.The nuclei distribution of ?asf1 was uneven,some parts more concentrating.Finally,the transcriptomes of wild type strains and the knockout transformants were sequenced and analyzed.It was found that the different expressed genes in WT and ?asf1 were 3023;the SEX and ?asf1 were 3716.The SEX and ?asf1,the WT and ?asf1,WT and SEX had the same 380 genes.Then those different expressed genes were analyzed.We concluded that there were 190 DEG involved in sexual development.And 10 key regulatory genes were initially defined.These results indicated that asf1 gene was necessary for the correct transcription of other genes.In conclusion,asf1 gene has significant impacts on the sexual and asexual development of S.eturmiunum through mediating histone assembly and disassembly that affects the expression of other genes.The acquisition of the knockout transformants of S.eturmiunum asf1 gene and its transcriptome analysis provide theoretical and empirical accumulation for the further study of the sexual development of Stemphylium.