辣椒黄化环斑病毒主要功能基因N、NSs、NSm的VIGS 体系构建及效率验证

Establishment and efficiency validation of VIGS systems for the main functional genes N, NSs and NSm of chilli yellow ringspot virus

关键词:  辣椒黄化环斑病毒，病毒诱导的基因沉默，实时荧光定量PCR，拷贝数，本氏烟，辣椒

Key words:  chilli yellow ringspot virus, VIGS, qRT-PCR, copy number, Nicotiana benthamiana  pepper

摘要:

辣椒黄化环斑病毒（chilli yellow ringspot virus, CYRSV）严重危害多种经济作物和园艺作物，其主要功能基因N、NSs、NSm与病毒侵染密切相关，构建N、NSs、NSm基因VIGS（Virus-induced gene silencing）体系以研究其致病功能的研究还未报道。为构建CYRSV N、NSs、NSm基因的VIGS沉默体系，分析N、NSs、NSm基因在CYRSV侵染中的作用，该研究将CYRSV N、NSs、NSm基因的300 bp左右靶片段分别构建到pTRV2沉默载体上，在不同接种模式下检测各基因的沉默效率，最后选择最佳接种模式再次在本氏烟和辣椒中进行沉默效率检测，应用绝对实时荧光定量PCR（qRT-PCR）方法检测N、NSs、NSm基因的拷贝数。结果显示，在本氏烟上N、NSs、NSm基因VIGS体系的沉默效率分别为82.07%、87.02%、94.39%；在辣椒上N、NSs、NSm基因VIGS体系的沉默效率分别为86.63%、89.22%、83.43%。该研究成功构建了CYRSV的N、NSs、NSm基因VIGS沉默体系，能高效抑制CYSRV侵染本氏烟和辣椒过程中N、NSs、NSm基因的拷贝数，抑制效率在82%和95%之间。该研究构建的N、NSs、NSm基因VIGS沉默体系能为后续研究CYRSV的致病机理提供前期材料，也为CYRSV抗病育种和田间绿色防控提供一定的理论依据。

Abstract:

Chilli yellow ringspot virus (CYRSV) causes severe diseases in many economically important plants such as cash crops and horticultural flora, its major functional genes (N，NSs，NSm) have closely relationship with viral infection, However, no report has described the construction of a VIGS system containing N、NSs、NSm genes to study its pathogenic function. To address this, a VIGS silencing system was constructed for the N, NSs and NSm genes of CYRSV, and the roles of these genes during CYRSV infection were analyzed. Approximately 300 bp target fragments of CYRSV N, NSs and NSm genes were respectively inserted into the pTRV2 silencing vector. The silencing efficiency of each gene was detected under different inoculation modes. The optimal inoculation method was selected and further validated in N.benthamiana and pepper plants. The copy number of N, NSs and NSm genes was quantified using absolute quantitative real-time PCR (qRT-PCR). The results showed that the silencing efficiency for VIGS system of N, NSs, NSm-VIGS system were 82.07%, 87.02%, and 94.39%, respectively, in N.benthamiana, and 86.63%, 89.22%, 83.43%, respectively, in pepper, compared with the control group. This study successfully established a VIGS system targeting the N, NSs and NSm genes of CYRSV, which effectively inhibit the copy number of N, NSs and NSm genes in N.benthamiana and pepper, with silencing efficiencies ranging from 82% to 95%. The N, NSs and NSm VIGS system developed in this study provides a valuable tool for future investigations into the pathogenesis of CYRSV and offers a theoretical basis for resistance breeding and environmentally friendly control strategies in the field.