| Soybean mosaic virus (SMV) is one of the major viral diseases of soybean(Glycine max (L.)Merr.) producing areas in the world. When soybean infects SMV, it will seriously affect the yield and quality. Breeding for disease resistance is the most cost-effective method of disease prevention and control SMV. They have screening the resources of resistance to SMV, and mapping the SMV gene. Currently, it has entered the functional analysis phase that the genetic researches fight against SMV. Virus induced gene silencing (VIGS) is a new technique for studying gene function, and it developed in recent years.In this study, we were to analyze the role of candidate genes for resistance to SMV by silencing the resistant candidate genes, analysis the changes of virus content and plant phenotypic. The main results were as follows:1. Cloning and analysis of soybean resistance candidate genes to SMV strains SC3Select the two genes which were named GmZ15 and GmR42 to analysis, based on the previous results of the positioning to SMV strains SC3 and according to the genes function annotations. According to published soybean genome sequence to design primers, two different genes cloned from Qihuang 1, PI96983, Zaol8 which were the resistant varieties of soybeans and Nannong1138-2 which was the susceptible variety out and compare the differences in gene sequences between resistant and susceptible varieties. The results showed that there were seven SNPs and two InDels for gene GmR42, sixteen SNPs and one InDel for gene GmZ15 between Nannong1138-2 and Qihuang 1. There were eight SNPs and one InDel for gene GmR42, sixteen SNPs and one InDel for gene GmZ15 between Nannong1138-2 and PI96983. There were seven SNPs and one InDel for gene GmR42, eight SNPs and two InDels for gene GmZ15 between Nannong1138-2 and Zao18. Further analysis of candidate genes expressed in the resistant line and susceptible line, the results showed that the two genes GmZ15 and GmR42 demonstrated relatively similar response mode showed a single peak in the two families, the expression of candidate genes at the same time point in the resistant line higher than the susceptible line. During Oh to 24h after inoculation, the expression of gene GmZ15 were increasing,and during 12h to 24h, it was rapidly increasing,and in 24h after inoculation it reached a peak. GmR42 had the same response pattern as GmZ15. However, the expression of gene GmR42 was increasing during Oh to 24h after inoculation. During 12h to 24h, it remained substantially constant.And it decreased during 24h to 48h after inoculation. These results indicated that the two candidate genes may be involved in the defense response.2. The verification of VIGS system based on BPMVTest the soybean leaves which inoculated the control vector by rubbing to confirm the changes of phenotype were caused by the intrusion of the empty vector.The expression of the gene GmPDS in leaves which inoculated with empty vector and inoculated the recombinant vector pGG7R2-GmPDS by fluorescence quantitative.The results showed that the expression of genes GmPDS which was inoculated with the recombinant viral vector significantly lower than the empty vector, it showed that the recombinant viral vectors pGG7R2-GmPDS was successfully invaded into soybean leaves, and successfully silenced the target gene GmPDS.Through these experiments, the method VIGS was described availability, and the candidate genes can be constructed the recombinant viral vector by empty vector.3. Researching the function of candidate genes by VIGSAccording to the method of the VIGS vector construction, the candidate genes GmZ15 and GmR42 inserted into viral vector on the basis of BPMV to construct the recombinant viral vectors pGG7R2-GmZ/5 and pGG7R2-GmR42.The changes expression of the target genes GmZ15 and GmR42 in soybean varieties PI96983 after inoculating the vectors pGG7R2, pGG7R2-GmZ15 and pGG7R2-GmR42 by fluorescence quantitative.The results showed that the expression of gene GmR42 which inoculated pGG7R2-GmR42 was lower than the vaccination empty vector, there was a difference of approximately 11-fold, and 4.1-fold difference of gene GmZ15. These results indicated that the two genes were subjected to different degrees of gene silencing.Test the virus content at the same time, and found the virus content which inoculated the pGG7R2-GmR42 and SC3 was 40 times higher than inoculated the pGG7R2 and SC3.The virus content which inoculated the pGG7R2-GmZ15 and SC3 was 3.5 times higher than inoculated the pGG7R2 and SC3. The trends of the candidate genes and viral content in Zao18 were consistent with PI96983. It described that SMV accumulated in plants when the candidate genes were silenced.The phenotype after genes were silenced in resistant and susceptible varieties identification results showed that the susceptible varieties Nannong 1138-2 and 8101 have symptoms after inoculating SMV seven days.However, the resistant varieties Qihuang No.1, Davis, Buffalo, Zao18, Kefeng No.1, Kwanggyo and Tiefeng25 appeared the symptoms when the two genes GmZ15 and GmR42 were silenced.lt had different incidence among different species, and it was between 80% and 100%. The symptoms which appeared in the resistant varieties were later than susceptible varieties.These results indicated that gene GmZ15 and GmR42 may participate in the process of soybean resistance to SMV, and the gene GmR42 may play an important role in the resistance process. |
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