Cloning And Functional Preliminary Study Of Resistance Gene To Soybean Mosaic Virus Strain Sc7 In Soybean

Soybean mosaic virus?SMV?causes one of the most prevalent virus diseases in many regions of the world where soybean?Glycine max?is grown,causing significant yield losses and seed quality deterioration.That breeding resistance varieties is the most effective and sustainable sound approach to control this disease.Plenty of studies on inheritance and molecular markers of resistance genes to SMV strains have been conducted in soybean.At present,the identification and distribution of strain groups of SMV in China has been accomplished by National Center for Soybean Improvement.But the resistance of the material corresponding to the key disease-resistant genes still need further excavation.SC7 is prevalent in the north of China,Huang Huaihai and the Yangtze River basin soybean producing areas of the strong strains.Soybean variety Qihuang No.l showed resistance to SC7 and other multiple SMV strains,and it was a relatively broad spectrum resistant germplasm.In this study,we selected the candidate gene selection interval between BARCSOYSSR₁3₁140-BARCSOYSSR₁3₁185 according to the reported resistance gene loci interval.We responded to the resistance gene analogue[NB-ARC domain-containing disease resistance protein?TIR-NBS-LRR class?family;sulfotransferase 2A;SERINE/THREONINE PROTEIN KINASE,etc.]selected 23 candidate genes for cloning and preliminary functional validation.Providing potential functional genes for transgenic resistance breeding.The main results were as follows:1.The SMV strain SC7 could not systematically infect the soybean variety Qihuang No.1.The results showed that there was no obvious viral disease in the phenotype of the inoculated leaves and the system leaves after Qihuang No.l inoculated with SMV for 3 weeks.Compared with Mock inoculation,No significant difference.Under the same conditions,the control varieties NN1138-2 were inoculated with SMV virus.The results showed that the contents of SMV in NN1138-2 and Qihuang No.l inoculated leaves and system leaves were detected by RT-PCR.The results showed that 30,35 and 40 PCR was used to detect the accumulation of SMV in the inoculated leaves of the control cultivar NN1138-2 and Qihuang No.1.However,the accumulation of SMV in the Non-inoculated leaves,30,35 and 40 PCR cycles was detected in NN1138-2,while the resistant strain Qihuang No.1 was not detected.The results showed that the SMV strain SC7 could not systematically infect the soybean variety Qihuang No.1.2.The overexpression gene qh108,qh92,qh104,qh111?Appendix 1?significantly enhanced the pathogenicity of SC7 to the inoculated leaves,whereas overexpression qh116,qh118 and qh122 mediated the resistance of the inoculated leaves to SC7.In this study,23 candidate genes in the Qihuang No.1 resistance locus were cloned and constructed into overexpression vector?pBINGFP2-Gene?.The gene was transiently expressed at 12 h,and after inoculation of SMV.The results showed that the SMV content of the inoculated leaves of overexpressing qh108,qh92,qh104 and qh111 was significantly increased.In contrast,the SMV content of the inoculated leaves of qh116,qh118 and qh122 was significantly reduced,and there was no difference with the positive control?overexpression cp?,indicating that qh116,qh118 and qh1422 mediate resistance to SC7.Overexpression of other genes did not significantly affect the accumulation of SC7 in the inoculated leaves.The above results suggest that overexpression of genes qh108,qh91,qh111,qh116,qh118 and qh122 promotes or inhibits the accumulation of virus SMV strain SC7 in the inoculation of native tobacco,suggesting that the above genes are soybean resistance to SMV breeding potential pathogenesis/Resistance gene.3.Overexpression qh119,qh121?Appendixl?were used to mediate the resistance of Pichia pastoris to pSMVGUS,but the overexpression gene qh112 increased the pathogenicity of pSMVGUS.In this study,23 candidate genes in the Qihuang No.1 resistant locus were cloned and constructed into overexpression vector?pBINGFP2-Gene?.The gene was transiently expressed in the leaves.After 12 h,the recombinant plasmid pSMVGUS?PBINGFP2?,the blue spots of the inoculated leaves of the inoculated leaves of qh119 and qh121 were significantly decreased,but not the same as the negative control?pBINGFP2?The positive control?overexpression of cp?was significant,qh119 and qh121 were involved in the resistance to pSMVGUS.In contrast,the blue spots of the inoculated leaves of overexpressing qh112 were significantly increased,indicating that the pSMVGUS content was increased.Overexpression of other genes did not significantly affect the accumulation of pSMVGUS in the inoculation of native tobacco.The above results suggest that the gene qh119 qh121 may inhibit the replication of the virus,gene qh112 is to promote the replication of the virus.Suggesting that the above genes are soybean resistance to SMV breeding potential resistance/pathogenic genes.