Sequence Analysis And Functional Verification Of Candidate Gene Cluster Mediated Resistance To Soybean Mosaic Virus

Soybean mosaic virus(SMV;genus Potyvirus)is one of the most destructive virus diseases in soybean and cause soybean mosaic and necrosis,which leads to yield loss and seed quality deficiency seriously.Mining resistance genes and analyzing the function of candidate genes are essential for resistance breeding.Fine mapping of Rsc4 conferring resistance to SMV has been conducted depth research in the world.Rsc4 were fine mapped on chromosome 14 in a 110-kb region by Nannong 1138-2 and Dabaima hybridization.Based on the fine mapping results of Rsc4,the amino acid sequences of three candidate genes in the region were aligned.To determine whether these three candidate genes were associated with Dabaima resistance to SMV,QRT-PCR and virus induced gene silencing(VIGS)were made for analysis genes expression and silencing,which lay the foundation for obtaining soybean resistant genes,resistance mechanisms,and transgenic disease-resistant varieties.The main results were as follows:1.Cloning and bioinformatics analysis of soybean resistance candidate genes Rsc4Previously Wang et al.2011 determined the candidate genomic region of Rsc4 in a 110-kb region.The corresponding region contains three complete nucleotide-binding site(NB)and leucine-rich repeat(LRR)type genes and one incomplete gene that is likely nonfunctional.In this study,three NB-LRR genes are considered as a candidate gene cluster to analyze their function.Three candidate genes(Rsc4-1,Rsc4-2 and Rsc4-3)were cloned from Dabaima(resistance cultivar)and Nannong1138-2(susceptible cultivar)which showed several SNPs of Rsc4-1 and Rsc4-2 between Dabaima and Nannong1138-2.Amino acid sequences analysis of three candidate genes in Dabaima indicated that the homology of Rsc4-1 with Rsc4-2 and Rsc4-3 were 79.89%and 81.24%separately and the homology of Rsc4-2 with Rsc4-3 was 84.76%.Phylogenetic analysis based on the amino acids sequence showed that the protein encoded by this gene had a close relationship with the reported R genes,such as wheat powdery mildew resistance gene PM3 and barley streak disease resistance genes Rdg2a.2.Resistant candidate genes expression analysisQRT-PCR analysis was used to examine the expression pattern of three genes post SMV-SC4 inoculation.Results from QRT-PCR demonstrated that the three genes were induced in SMV infected plants and transcript abundance differed significantly between resistant and susceptible plants.In the resistant soybean cultivar,except for 12 hpi,comparing with Ohpi,the relative expression of all three genes up-regulated at other time points and then kept stable.In the susceptible soybean cultivar,expect for 12hpi the relative expression of Rsc4-1 and Rsc4-2 up-regulated,comparing with Ohpi,the relative expression of these three genes all down-regulated at other time points and kept stable finally.These results support the hypothesis that Rsc4-1,Rsc4-2 and Rsc4-3 are involved in soybean resistance to SMV..3.Analysis the function of candidate genes by virus induced gene silencingAccording to the nucleotide sequences of Rsc4-1,Rsc4-2 and Rsc4-3 in Dabaima,a 459 bp fragment conserved among the three genes was inserted into the modified BPMV-pGG7R2 vector.After Dabaima inoculated with the recombinant silencing vector BPMV-Rsc4,double antibody sandwich enzyme-linked immunosorbent analysis(DAS-ELISA)and QRT-PCR results indicated that Dabaima can be infected with BPMV-Rsc4 and RNA accumulation for the Rsc4 gene cluster was down-regulated 78%compared to the BPMV empty vector control(BPMV-0).Subsequently,Rsc4-1,Rsc4-2 and Rsc4-3 transcript accumulation was detected separately using gene-specific primers.Results indicated the accumulation of Rsc4-1,Rsc4-2 and Rsc4-3 mRNAs were down-regulate 93%,42%and 88%,respectively,in plants inoculated with BPMV-Rsc4compared to BPMV-0.To evaluate the effects of gene silencing on SMV resistance,we inoculated the silenced Dabaima plants with SMV.QRT-PCR results denominated that the transcript accumulation of SMV-CP gene up-regulated significantly comparing with control on the SMV inoculated leaves.SMV accumulation on inoculated leaves were 39 times,2.5 times and 346 times higher than control at 3d,6d,9d.Silenced plants systemic leaves developed mosaic symptoms and stunned,although the disease symptoms were severe than those induced by BPMV-0(control).In addition,silenced plants are abnormal and clear disease symptoms top necrosis puckered along the veins and curled downward were seen in systemic leaves.Overall,we found that mixed infection between BPMV and SMV occurred in silenced plants(BPMV-Rsc4)but not in BPMV-O.Further confirmation we evaluated the accumulation SMV RNA in inoculated and upper systemic leaves by RT-PCR and DAS-ELISA.Results were consistent with phenotypic results,the accumulation of SMV RNA was readily detectable in both inoculated and systemic leaves in silenced plants.Collectively all the results suggests that down-regulation of Rsc4 gene family inducing the susceptibility in Dabaima.