Cyclin-dependent Protein Kinase PsCdc2 Of Phytophthora Sojae And Its Interaction With Borrelidin

Phytophthora root rot disease,casued by P.sojae,is one of seriously worldwide diseases.In China,soybean root rot causes significant economic losses every year.However,macrolide antibiotics borrelidin has very high inhibitory activity against P.sojae which may become a new type of highly effective agricultural antibiotic.The antifungal activity of borrelidin against P.sojae was mediated by inhibition of threonyl-t RNA synthetase(Thr RS),which is formating Thr RS-borrelidin complex to inhibit the protein synthesis in previous research.The purpose of this study was to explore the other ways of borrelidin against P.sojae.In this study,bioinformatics methods are used to analyze the cyclin-dependent protein kinase gene which was homologous to yeast Cdc28,the inhibition target of in borrelidin the cell cycle.The genetic engineering technology was used to clone psCdc2 gene of P.sojae and overexpressed in E.coli and yeast to obtain purified protein.The interaction of borrelidin and psCdc2 was analyzed by fluorescence spectroscopy.Effect of psCdc2 gene silencing mediated by dsRNA on P.sojae was studied.The results were as follows:(1)Bioinformatics analysis of cyclin-dependent protein kinase psCdc2 gene showed that the protein consists of 20 amino acids,the molecular weight 35,478 Da,and is a stable protein with the isoelectric point of 8.56,no signal peptide,and it is an ?/? protein.Ten proteins interacting with the yeast Cdc28 gene all were found their homologous proteins in the genome of P.sojae,and the identity of proteins is from 26% to 46%,indicating that protein interaction networks in P.sojae is similar to yeast.(2)The psCdc2 gene was obtained by reverse transcription PCR and cloned into E.coli pET 32a(+)expression vector by gene engineering technology.The recombinant plasmid was transformed into BL21 for expression.psCdc2 were purified by inducing in low temperature and dialysis after denaturation and renaturation.However,for the forming of inclusion bodies of the expressed protein,it was cloned into the pPIC9 K expression vector of Pichia pastoris,and transformed into P.pastoris by electroporation,and purified by nickel column to obtain soluble cyclin-dependent protein kinase psCdc2 of P.sojae.(3)Fluorescence spectroscopy analysis was used to study the interaction between borrelidin and the cyclin-dependent protein kinase psCdc2.Results showed that with the increase of the concentration of borrelidin,the fluorescence intensity of the protein decreases continuously which indicated that borrelidin and ps Cdc2 interacted.While the quenching constant Ksv increased with temperature which suggested that the fluorescence quenching of psCdc2 by borrelidin is dynamic quenching,and the interaction between borrelidin and cyclin-dependent protein kinase psCdc2 is caused by collision.(4)The dsRNA of psCdc2 was synthetized in vitro,and transformed to protoplast of P.sojae mediated by PEG.The dsRNA mediated psCdc2 gene silencing in P.sojae did not significantly change the colony morphology of the psCdc2 transformants on the regeneration medium.However,the growth rate of the strains slowed down and the number of induced sporangia decreased and was abnormal.Those results indicated that the silencing of the cyclin-dependent protein kinase psCdc2 gene affects the growth of P.sojae.Our study suggested that the interaction between borrelidin and cyclin-dependent protein kinase psCdc2 is caused by collision,and the silencing of the cyclin-dependent protein kinase psCdc2 gene affects the growth and sporangia development of P.sojae.