Clone And Functional Analysis Of Succinate Dehydrogenase Gene PsSDHA In Phytophthora Sojae

The plant pathogen Phytophthora sojae is one of the most destructive diseases that could cause severe loss in soybeanl production.It is one of the most notorious pathogens which caused tremendous economic loss in soybean production annually.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,RNAi has become a commonly used and effective method in Phytophthora sojae functional gene analysis.Succinate dehydrogenase(SDH),is the key enzyme of TCA cycle,which is the only one embedded in the membrane of many subunits enzyme in the TCA cycle.It plays an important role,which is a hub connecting oxidative phosphorylation and electron transfer,providing the aerobic capacity and respiratory chain of the organism.Succinate dehydrogenase is a kind of complex,which subunit A contains a covalently linked to FAD binding sites,succinic acid,coenzyme Q.Subunit B contains three iron sulfur group: 2Fe-2s,3Fe-4s,4Fe-4s.The activity of succinate dehydrogenase can be used as indicators of the degree of the three operation which is a key enzyme in tricarboxylic acid cycle.TCA cycle provides the energy for the growth of Phytophthora sojae.In order to research the roles of SDHA gene in the phytophthora sojae,at first we had cloned the conservative PsSDHA gene to construct the PsSDHA silenced expression vector named PHAM34-PsSDHA,and then utilized PEG to mediate the P.sojae protoplasts transformation experiment.Throughout screening the transformations,we got silenced mutants A1 and A3 identified by PCR and qRT-PCR,which have the good suppressed effect.We also did some experiences about the phenotypic analysis of the silenced mutants.In the 10% V8 culture medium under culture conditions,the growth of mutants had significant difference with the wild strain or the controlled strain.Pattern of wild strains mycelium is slender,less bifurcate while the silenced mutants strains is short,more bifurcate.However,the silenced mutants growth were obviously inhibited in the 10% V8 culture medium containing 0.7M NaCl,0.01%SDS,3mMH2O2,200ng/mL Congo Red.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.