| 摘要: |
| 辣椒黄化环斑病毒(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基因病毒诱导的基因沉默(VIGS)体系以研究其致病功能尚未有报道。为构建CYRSV N、NSs、NSm基因的VIGS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,并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。结果表明:(1)在本氏烟上,N、NSs、NSm基因的VIGS体系的沉默效率分别为82.07%、87.02%、95.39%; 在辣椒上,N、NSs、NSm基因的VIGS体系的沉默效率分别为86.63%、89.22%、83.53%。(2)成功构建了CYRSV N、NSs、NSm基因的VIGS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,沉默效率在82%至95%之间。该研究构建的N、NSs、NSm基因的VIGS体系为后续研究CYRSV的致病机理提供了前期材料,也为CYRSV抗病育种和田间绿色防控提供了一定的理论依据。 |
| 关键词: 辣椒黄化环斑病毒(CYRSV), 病毒诱导的基因沉默(VIGS), 实时荧光定量PCR, 拷贝数, 本氏烟, 辣椒 |
| DOI：10.11931/guihaia.gxzw202502013 |
| 分类号:Q953 |
| 文章编号:1000-3152(2026)05-0802-12 |
| Fund project:国家自然科学基金(32360638); 云南省科技计划项目基础研究专项青年项目(202301AU070121); 云南省科技计划项目基础研究专项面上项目(202501AT070087); 云南省科技计划项目基础研究专项重大项目(202501BC070017); 云南省种子种业联合实验室项目(202205AR070001)。 |
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| Establishment and efficiency validation of VIGS systems for the main functional genes N, NSs and NSm of chilli yellow ringspot virus |
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LI Yu1, CHEN Yongdui1, WU Kuo1, MA Chuanzhi2,
ZHANG Jie1*, ZHANG Zhongkai1
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1. Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences/Yunnan Province Key
Lab of Agricultural Biotechnology, Kunming 650205, China;2. College of Agriculture and
Biotechnology, Yunnan Agricultural University, Kunming 650500, China
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| 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 virus-induced gene silencing(VIGS)system containing N, NSs, NSm genes to study its pathogenic function. To address this, a VIGS 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 Nicotiana 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 were as follows:(1)The silencing efficiencies for VIGS system of N, NSs, NSm were 82.07%, 87.02%, and 95.39% in N.benthamiana, respectively, and 86.63%, 89.22%, 83.53% in pepper, respectively, compared with the control group.(2)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. |
| Key words: chilli yellow ringspot virus(CYRSV), virus-induced gene silencing(VIGS), qRT-PCR, copy number, Nicotiana benthamiana, pepper |
