Identification Of Candidate Genes Of Phytophthora Sojae Resistance Gene RpsJS

As an important industrial crop,soybean(Glycine max(L.)Merr.)is a main source of vegetable protein and oil for humans and animals.Major biotic stresses such as series of pathogens have major implications to the quality and yield of the crop plants worldwide.Phytophthora root and stem rot caused by Phytophthora sojae is one of the most destructive soybean diseases.The effective way to control the disease is breeding with dominant Rps(Resistance to Phytophthora sojae)genes.Due to the selection pressure,P.sojae has highly frequence of gentic variation and cause the emergence of newly virulence stains.Till now,there are more than 200 isolates have been reported.Hence,the exploration of new resistant genes is not only contributes to resistance breeding but also improving the genetic basis of soybean varieties.At present,33 genes against P.sojae Rps have been identified and located on 9 chromosomes of soybean.Only Rps1k has been cloned,and its resistance mechanism to P.sojae pathogens is not clear.Previous studies have found that the soybean variety nannong 10-1 is completely resistant to the physiological races of P.sojae JS12.Resistance gene derivation indicated that nannong 10-1 might contain new Rps genes;Genetic analysis of resistance showed that resistance of nannong 10-1 to P.sojae JS12 was controlled by a dominant single gene,and call it RpsJS;RpsJS was located on chromosome 18 of soybean G linkage group.In the early stage,the laboratory obtained 75 Kb fragments by genomic resequencing and sequence splicing with molecular markers,Eight NB-LRR proteins were predicted to be candidate resistance genes on this fragment.In this study,we first cloned 8 candidate disease-resistant genes.In order to screen and identify functional disease-resistance genes,8 candidate genes were over-expressed in soybean Williams varieties by agrobacterium-mediated soybean genetic transformation technology.Furthermore,Transgenic hairy roots were screened for disease resistance function by disease-resistance level,gene expression and disease-resistance correlation and infection cytology observation.The main results are as follows:1.The ratio of disease-resistant phenotype of RpsJS3 soybean hairy roots was significantly higher than that of other genesPlant expression vectors of 8 candidate disease-resistant genes were constructed with native promoter and 35s promoter respectively.And the transgenic hairy roots were obtained through soybean hairy root transformation mediated by Agrobacterium Rhizogenes K599.The obtained transgenic hairy roots were inoculated with P.sojae JS12.HR disease-resistant phenotype was found in the hairy root transfection of RpsJS3 and RpsJS5.The ratio of HR disease-resistant phenotypes in RpsJS3 hairy roots was respectively(self-promoter:5.6%,35s promoter:7.2%).The ratio of HR disease-resistant phenotypes in RpsJS5 hairy roots was respectively(self-promoter:1.8%,35s promoter:3.1%).2.The expression level of RpsJS3 in disease-resistant hairy root tissues was significantly higher than that in susceptible tissues,which was positively correlated with the disease-resistant phenotype.Quantitative PCR was used to verify the gene expression level of transgenic hairy roots with different phenotypes 48h after inoculation,It was found that the gene expression level of RpsJS3 in disease-resistant samples was significantly higher than that of susceptible samples There was no significant difference in gene expression level between RpsJS5 disease-resistant samples and disease-susceptible samples;The candidate disease-resistant gene RpsJS1、RpsJS2、RpsJS4、RpsJS6、RpsJS7 and RpsJS8 showed no disease-resistant phenotype after the transgenic hairy root was inoculated with JS12.And overexpression of candidate genes was detected in the hairy roots of the susceptible phenotypes.A comprehensive analysis of the correlation between gene expression level and phenotype showed that the expression level RpsJS3 in disease-resistant hairy root tissue was significantly higher than that in disease-resistant tissue,which was positively correlated with disease-resistant phenotype.It is believed that RpsJS3 may be a gene with disease-resistance function.3.Overexpressed RpsJS3 transgenic soybean hairy roots showed a significantly higher proportion of disease-resistant phenotype hairy roots than other genes.The overexpressed RpsJS transgenic hairy root by inoculation with P.sojae JS12.According to the statistical phenotypic results,The inoculation phenotypic trend was basically consistent with the preliminary screening results.It was found that RpsJS3 positive hairy roots were overexpressed after inoculation with P.sojae JS12,the incidence of root hair was mild,and HR phenotype was observed in 40%of hairy roots that have been inoculated.The ratio of HR disease-resistant phenotype in hairy roots was significantly higher than that in other genes and negative controls.We think RpsJS3 may have the function of resistance to P.sojae JS12.4.Single cell necrosis phenotype was observed after overexpressed RpsJS3 soybean hairy roots were inoculated with zoospores.of P.sojae.Overexpressed transgenic hairy roots were infected with zoospores of P.sojae,according to the observation of infection,it was found that the phenotype of single cell necrosis appeared in the hairy roots of overexpressing RpsJS3 after zoospore infection.The phenotype of hyphae elongation occurred in the hairy root of overexpressed others genes and the control group.In summary,cytological observations show that RpsJS3 had the function of resisting P.sojae JS12.