Cloning and Functional Analysis of GmRHF1 Gene Against Soybean Mosaic Virus
[Objective]Soybean mosaic virus (SMV) disease causes by SMV seriously restricts soybean yields and qualities.In this study,we cloned GmRHF1,which encodes a Ring-H2 type zinc finger protein (RHF),and identified its function in SMV resistance through the sequence variation analysis and the virus induced gene silencing (VIGS) assay.Yeast two-hybrid and luciferase complementation assay (LCA) were used to screen and verify the interacting proteins of GmRHF1.The research lay a foundation for further exploring the mechanism of GmRHF1 in soybean resistance to SMV.[Method]Firstly,GmRHF1 was cloned from soybean varieties of Xudou 14 (highly resistant to SMV) and Shishanheidou (highly susceptible to SMV),and the consistency of sequence variation was then analyzed in representative resistant and susceptible soybean resources,so as to excavate the excellent alleles with SMV resistance.Secondly,the expression abundance of GmRHF1 in different soybean tissues and under SMV treatment were analyzed by real-time quantitative PCR (RT-qPCR),respectively.At the same time,the function of GmRHF1 in SMV resistance was identified by the VIGS assay.The interacting proteins of GmRHF1 were screened and verified by yeast cDNA library screening,point to point verification and LCA experiments.Finally,the function of GmClpP6 on SMV resistance was verified,and the biological significance based on the interaction of GmRHF1 and GmClpP6 for soybean resistance to SMV was clarified.[Result]GmRHF1 contained a conserved RING-H2 domain of the E3 ubiquitin protein ligase,and belongs to the typical RING-H2 finger family.The results of sequence variation analysis showed that a natural non-synonymous mutation existed in the CDS region of GmRHF1 among various soybean varieties,providing a probable relevance to the SMV resistance or sensibility.After SMV inoculations,the expression level of GmRHF1 in resistant soybean was significantly higher than that in susceptible material by RT-qPCR assay.Through the VIGS experiments,GmRHF1 was effectively silenced and the soybean leaves showed a weakened SMV resistance than the normal plants,indicating that GmRHF1 could enhance soybean resistance to SMV.Based on the yeast cDNA library,30 potential interacting proteins of GmRHF1 were screened.The interaction between GmRHF1 and GmClpP6 was confirmed by the two-hybrid and LCA experiments.Furthermore,the soybean resistance to SMV was also reduced in soybean leaves with the silenced GmClpP6 by the VIGS experiments.[Conclusion]The non-synonymous mutation identified in the coding sequence of GmRHF1 may be a key SNP variation related to the SMV resistance and sensibility;and GmRHF1 can interact with GmClpP6.GmRHF1 plays an important role in SMV resistance.
万方数据
维普
