Analysis Of Verticillium Dahliae U6 Promoter And Scopulariopsis Gossvpii RDNA

Verticillium wilt is one of the main diseases of cotton,which has caused serious losses to cotton production.In China,the main pathogen causing verticillium wilt of cotton is Verticillium dahliae.With the development of gene editing technology,the CRISPR/Cas 9 system has been applied to fungal gene function research.In the CRISPR/Cas 9 system,the U6 promoters are usually used as elements of sgRNA transcription in different organisms but its length in fungi is not yet clear.The purpose of this study was to analyze the length of endogenous U6 promoter so as to provide a reference for constructing a V.dahliae CRISPR/Cas 9 systemScopulariopsis gossypii is the only pathogen infecting plants found in the genus Scopulariopsis so far.The original study found that the universal primers could not amplify the ITS region for this species,which affected the taxonomic study.In this study,the rDNA sequence obtained from PacBio single-molecule long-read sequencing was analyze and the reason why the universal primer could not be amplified was revealed.In addition,the role of the ITS region in the taxonomy of Scopulariopsis was evaluated1.Study on U6 promoter of Verticillium dahliaeIn order to analyze the length of U6 promoter,four knockout vectors with different lengths of V.dahliae U6 promoter were constructed by one-step cloning method and they were transferred into protoplasts by PEG-mediate method.The mutants with different lengths of U6 promoter were screened on the selectively resistant PDA and identified by PCR amplificationIn order to reveal the effect of U6 promoters with different lengths on U6 snRNA expression,four representative mutants ?VdU6-5P(527bp),?VdU6-4P(423bp),?VdU6-3P(323bp)and ?VdU6-2P(230bp)were selected and expression of U6 snRNA in four mutants were detected by qRT-PCR analysis.The detected results showed that the relative expression level of mutants ?VdU6-5P,?VdU6-4P,?VdU6-3P,and ?VdU6-2P was 0.54,0.44,0.43 and 0.43,respectively,comparing with the wild-type isolates,showing that there was not significant difference among mutants.Therfore,the U6 promoter with 230 bp length in V.dahliae may be used as elements of sgRNA transcription in CRISPR/Cas 9 system2.Study on the ribosomal DNA sequence of S.gossypiiBased on the results of PacBio single-molecule long-read sequencing of S.gossypii,a 2578 bp rDNA sequence was obtained.The blast search in NCBI database showed that rDNA sequence of S.gossypii was 97%identitical to that of S.brevicaulis.The incompletely identical bases in binding site of nine universal primers in the SSU region of rDNA sequence of S.gossypii were found by comparison with universal primers and the base mutation ranged from 2 to 5 bp.On this basis,nine universal primers pares were revised according to change of the bases,and subsequently,the modified universal primers were verified by PCR amplificationIn order to evaluate the role of ITS in fungal taxonomy,phylogenetic trees with four-loci sequences(ITS,LSU,TEF1 and TUB)and with three-loci sequences(LSU,TEF1 and TUB)dataset were constructed with maximum likelihood respectively Phylogenetic analysis showed that although different species on phylogenetic tree with the three-loci(not ITS)dataset could be distinguished clearly,intraspecific relationship among different taxa could be revealed within the same species on phylogenetic tree with the four-loci dataset.This shows that the sequence of ITS region plays an important role in taxonomy of Scopulariopsis.