Clone And Functional Analysis Of Histone Acetyltransferase Gene PsGCN5in Phytophthora Sojae

The plant pathogen Phytophthora sojae is one of the most destructive diseases that could cause severe loss in soybeanl production. In recent years,with the improvement of Phytophthora sojae system and Phytophthora sojae genome sequencing project, Phytophthora sojae has become the model strain for people to analyse the oomycete gene,the RNAi has become a commonly used and effective method in Phytophthora sojae functional gene analysis.Histone acetyltransferase (HATs) is the important essential of organisms. It could control the overall level of organism genome by interact with the HDAC(Histone Deacetylase), and they mediate the expression of different genes by regulating histone H3, H4core histones Lys-residues of acetylation and deacetylation. At present, the GCN5gene is the kind of regulatory genes and the representative of the HATs family. There is highly homology about the amino acid residues encoded by the GCN5gene, especially, the protein domains and the histone lysine residues binding site in the345amino acid residues, the homology is more than97%in Phytophthora, but its amino acid sequence between different species have obvious difference.After analysing the PsGCN5expression in the Psojae different asexual life stages and the infection phases by qRT-PCR, PsGCNS expression relative to the mycelial stage is down-regulated in the asexual life stages, however, there is a certain degree of increase in the post-infection stages, at the time of the infection for12H, its expression is up-regulated obviously and about2.8times as much as the mycelial stage, so we could consider that the PsGCNSplays a positive role in the P.sojae post-infection stages.In order to research the roles of GCN5gene in the phytophthora sojae, at first we had reproducted the conservative PsGCNS gene to format the PsGCN5silenced expression vector named pHAM34-PSGCN5, and then utilized PEG to mediate the P.sojae transformation experiment. Throughout screening the transformations, we got three silenced mutants named T37, T66, T68identified by PCR and qRT-PCR, which have the good suppressed effect in the silenced mutants by qRT-PCR.We also did some experiences about the phenotypic analysis of the silenced mutants. In the10%V8culture medium under culture conditions, the growth of mutants had no significant difference with the wild strain or the controlled strain. There is no difference about the silenced mutants zoospore or Cyst germination rate, and the oospore production of the silenced mutants is not effected, too. However, the silenced mutants growth were obviously inhibited in the10%V8culture medium containing5mM H2O2,0.01%SDS and0.7M NaCl. With the result of infection experiment in vivo and in vitro, the pathogenicity of silenced mutants has significantly reduced compared with the wild strain and the controlled strain.