Functional Analysis Of Soybean Mosaic Virus Resistance Gene

Soybean mosaic virus(SMV)disease is one of the prevalent diseases of soybean,which has brought huge losses to Chinese soybean production.Soybean disease resistance breeding is currently the most cost-effective measure to control soybean mosaic disease,and the discovery of disease resistance genes has become the basis of disease resistance breeding.At present,the screening of SMV resistance germplasm and the location of SMV resistance genes have been deeply studied.Based on the localization of soybean SMV resistance strain SC3 gene Rsc-pm in this laboratory,two genes with TIR-NBS-LRR typical disease resistance domain(GmR47,GmR51)were cloned and the functions of these two genes were analyzed.The main contents of this study are as follows:1.Cloning and analysis of soybean SC3 resistance candidate geneBased on the localization of soybean SMV strain SC3 resistance gene in the early stage of the laboratory,the candidate gene function was predicted and screened in the localization interval,and finally two TIR-NBS-LRR typical disease resistance structures gene with highly homologous were selected.Candidate gene clone primer were designed based on the published soybean genome data,it was cloned from the resistant variety PI96983,Qihuang No.1,and the susceptible variety Nannong 1138-2.The result of Nucleic acid sequence alignment showed that GmR47 had 8 SNPs in the resistant variety PI96983 and the susceptible variety Nannong 1138-2,which resulted in mutations in 5 amino acids.GmR51 contained 8 SNPs among the resistant varieties,which resulted in mutations in 3 amino acids.GmR47 has 5 SNPs in the resistant varieties Qihuang 1 and Nannong 1138-2,which resulted in mutations in 3 amino acids.GmR51 has 10 SNPs in the two varieties,which resulted in mutations in 4 amino acids.Bioinformatics analysis showed that both GmR47 and GmR51 genes have mutations in amino acid sites in resistant varieties,and the mutation sites are located in conserved domains,which may be an important factor leading to different functional functions of candidate genes in resistant varieties.At the same time,the functional prediction results of these two genes encoded proteins were tobacco mosaic virus(TMV)resistant N protein;the homologous ratio between species showed that GmR47 and GmR51 genes were close to wild soybean.2.Analysis of the expression levels of soybean SC3 resistance candidate genes and their alternative splicingUsing qRT-PCR technology,the expression levels of GmR47 and GmR51 in resistant varieties after virus induction were analyzed.The results showed that GmR47 and GmR51 could increase the expression level in response to SMV infection and the expression level in resistant varieties higher than susceptible varieties.GmR47 and GmR51 were found to have different splice variants.The response of different splices to SMV indicated that all splices could increase expression in response to virus induction and thier were confer resistant to SMV.The expression level of the cultivar was higher than that of the susceptible variety.The expression of the variable splicing IN1 was more obvious over time,and the expression levels of IN2 and IN3 were relatively stable,indicating that these splicing bodies may participate in the resistance of soybean to SMV.These results indicated that both GmR47 and GmR51 genes and their alternative splicing can respond to virus induction and increase expression,suggesting that these two genes may be involved in the resistance of resistant varieties to SC3.3.Using VIGS to study the function of soybean SC3 resistance candidate genesIn this experiment,we successfully silenced the SC3 resistance candidate genes GmR47 and GmR51 by the VIGS silencing system based on BPMV(Bean Pod Mosaic Virus).After inoculation of the candidate gene silencing vector pBPMV-GMR,the expression of candidate genes in the plant was significantly lower than that in the control,the candidate gene was shown to be significantly silenced.At the same time,the silencing plants showed symptoms such as foliar curling and dwarfing of the plants about one week after inoculation,and the top necrosis occurred within 6 weeks after inoculation,which eventually led to the death of the whole plant.The change in phenotype after silencing of the candidate gene preliminarily speculated that this candidate gene may be involved in the extreme resistance reaction of plants.In order to verify the change of candidate gene silencing and subsequent plant resistance,SC3 was inoculated on the first pair of compound leaves that were developed 7 days after the silencing vector was inoculated,and the virus content in the silenced plants was detected by real-time quantitative quantification(qRT-PCR).The results showed that the virus content in the leaves of SC3-inoculated leaves was significantly increased within 6,9,and 13 days after SC3 inoculation,it is preliminarily proved that the candidate genes GmR47 and GmR51 are involved in the resistance of P196983 to SMV.4.Resistance identification of candidate knockout varieties and screening of hybrid progenyUsing the CRISPR-Cas9 system to knocked out the SC3 resistance candidate genes GmR47 and GmR51 in the williams82 variety,the resistance analysis of the gene knockout T4 generation CR-26 was performed.The results showed that the symptoms of CR-26 were more serious than wild type Wiliams82 after SC3 inoculation.Hybridization of CR-26 with soybean SC3 resistance variety Qihuang No.l and Dabaima was carried out,the F3 plants whose resistance genes were homozygous and whose candidate genes were edited by the CRISPR-CAS9 system were used for resistance identification.After the candidate gene was edited,most of the resistant homozygous offspring of the hybrid progeny showed symptoms of SC3;however,a small number plants remained resistant to SC3 after the candidate gene was edited.It is presumed that GmR47 and GmR51 are disease-resistant genes.Some individual plants with no symptom after SC3 inoculation,needs to find the reason.