Clone And Functional Analysis Of Fumarate Reductase Gene PsFRD In Phytophthora Sojae

With the increase of global soybean production,the reports of soybean diseases caused by Phytophthora sojae.Phytophthora sojae is a destructive disease of soybean,it can lead to a great loss of soybean production,the disease as early as reported in the United States in 1948,and then quickly become the one of the important diseases which can affect the growth of soybean.Fumarate reductase;FRD plays the role of anaerobic energy metabolism in mitochondria including lower eukaryotes and bionts survived in micro oxygen or anaerobic environment.It can bind to the complex?called flavin adenine dinucleotide prosthetic group?FAD?in mitochondria,and restore Fumaric acid to succinic acid,it is the adverse reaction of succinate dehydrogenase,which is an important component of the tricarboxylic acid cycle.This dissertation mainly reasearched the cloning and function analisis of fumarate reductase gene PsFRD in Phytophthora sojae.Try to reveal the relationship between PsFRD and phytophthora sojae pathogenic mechanism,and provide theory basis for the targets of the new efficient biological pesticide.Getting the following conclusions:?1?Using FRD sequences reported in Phytophthora infestans for sequence alignment,getting phytophthora sojae PsFRD gene sequence.The amino acid sequence of PsFRD protein structure prediction shows that it contains a FAD_binding₂ conservative domain structure.According to the system of evolutionary tree,the PsFRD has a tie relationship to parasitic Saprolegnia parasitica and phanomyces astaci,and distant relatives to terrestrial plants such as Arabidopsis thaliana.ExPASy online website,found that the protein prediction of PsFRD is a kind of hydrophilic proteins,with stable structure and no across the membrane structure.?2?Using the technolog of RT-PCR,testing the expression of gene PsFRD in diffferent growing stages of phytophthora sojae.Found that the expression of gene PsFRD in sporangium and zoosporangium are lower than mycelium and geiminated cyst,the highest expression of gene PsFRD is 1.58 after infection 24 h.?3?We Successfully build PsFRD-PTOR-GFP transient expression vector,and get 5 mutant strains.Selecting mutants PsFRD expression in relative to the amount of p6497 express 0.3 and 0.5 times the amount of N3,N5 do subsequent experiment.?4?We test the adversity stress resistance on N3,N6.the adversity stress resistance in 3 nmol/L H2O2V8 medium N3,N6 strains in five days of hypha growth length into 1.79 cm and 2.38 cm is 39.87% and 53% of the wild type.In 0.7 mol/LNacl medium N3,N6 strains hypha growth length of the 5 days can be divided into: 1.83 cm and 2.12 cm is 63.76% and 73.87% of the wild type WT 2.87 cm.The experiment shows that ability of salt resistance ang reactive oxygen resistance of mutants N3,N6 declined.?5?We test the virulence of the PsFRD silence mutants,including mutants infect the leaves of the susceptible varieties of hefeng 47 and hypocotyl inoculation in vivo strain experiment.We find that the mutant regardless of in vitro leaf or in vivo strain are displayed weaker pathogenicity compared to the wild type P6497,performed that the spot area percentage of the total leaf in N3,N6 are about: 2.07% and 2.81%,while 62.88% of WT disease spot percentage after phytophthora strains N3 N6 WT infecting the vitro leaf of soybean 60 hours later.