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

Soybean blight caused by Phytophthora sojae is one of the most destructive diseases that could lead to severe loss in soybeanl production.P.sojae mainly achieve the goal that infect the host through secreting effect factors,such as necrosis protein.So if we can hinder the process,we can effectively reduce the production and secretion of related molecules,we can effectively control the disease as well.In this dissertation,the succinate dehydrogenase gene PsSDHB was cloned successfully,and the function of the gene was analysed in order to reveal the power-providing link of P.sojae and the synthetic and secretory mechaism of the related effectors,and to provide theory basis for the devision of molicular target of new fungcide controlling the disease.The main results are as follows:(1)we first cloned the conservative PsSDHB gene to construct the PsSDHB silenced expression vector pTOR-GFP-PsSDHB,and then utilized PEG to mediate the P.sojae protoplast transformation experiment.Through transformation screening,we obtained the silenced mutants B1 and B2 identified by PCR and qRT-PCR,which have good silenced suppression effects.(2)By BLAST,the domain and protein sequences of PsSDHB amino acid residues were found first,and the unrooted neighbor-joining phylogenetic tree of normal model organisms was built,in which PsSDHB was placed in a clade with a homolog from P.infestans.(3)qRT-PCR analysis results showed that the gene was not only expressed during various life stages but also during the course of infection,with the highest expression levels observed 12 h after infection.The expression levels of the gene were low at zoosporangium stage,zoospore stage,with the lowest at cystospore stage.(4)The phenotypes of the mutant strains were annalysed.The results showed that the growth rates of the silenced mutant strains were obviously slower than that of the wildtype.On 10% V8 medium containing 0.7 M sodium chloride(NaCl),0.01%sodium dodecyl sulfate(SDS),3 mM hydrogen peroxide(H2O2)and 200 ng/mL Congo Red,the mycelium growth of the silenced mutants was clearly inhibited,with some growing slowly and some not growing.The pathogenicity tests indicated that the pathogenicity of the silenced mutants was significantly reduced compared with the wild-type strain and the control strain.