病毒载体介导的基因沉默体系对朱顶红褪绿环斑病毒NSm基因的沉默效应  

Silencing effect of a virus vector-mediated gene silencing system on the gene NSm of Hippeastrum chlorotic ringspot virus

作　　者：许云玉 李浩 陈敏 贾志强 高雪[5] 陈增敏 赵于丁 刘雅婷[1] 李永忠[6] Xu Yunyu;Li Hao;Chen Min;Jia Zhiqiang;Gao Xue;Chen Zengmin;Zhao Yuding;Liu Yating;Li Yongzhong(College of Plant Protection,Yunnan Agricultural University,Kunming 650201,Yunnan Province,China;Ning’er County Branch,Pu’er City Company of Yunnan Tobacco Company,Pu’er 665199,Yunnan Province,China;Yimen County Branch,Yuxi Tobacco Company,Yuxi 651100,Yunnan Province,China;Yunnan Province Protection and Quarantine Station of Zhangtong City,Zhaotong 657000,Yunnan Province,China;College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,Yunnan Province,China;College of Tobacco,Yunnan Agricultural University,Kunming 650201,Yunnan Province,China)

机构地区：[1]云南农业大学植物保护学院,昆明650201 [2]云南省烟草公司普洱市公司宁洱县分公司,普洱665199 [3]玉溪市烟草公司易门县分公司,云南玉溪651100 [4]云南省昭通市植保植检站,昭通657000 [5]云南农业大学农学与生物技术学院,昆明650201 [6]云南农业大学烟草学院,昆明650201

出　　处：《植物保护学报》2023年第4期923-931,共9页Journal of Plant Protection

基　　金：国家自然科学基金(32260681);国家重点研发计划(2021YFD1400200)。

摘　　要：为探讨烟草脆裂病毒(tobacco rattle virus,TRV)载体介导的基因沉默技术对朱顶红褪绿环斑病毒(Hippeastrum chlorotic ringspot virus,HCRV)运动蛋白基因NSm的沉默效应,以本氏烟Nicotiana tabacum为材料,构建靶向HCRV NSm序列的基因沉默表达载体pTRV2-NSm,经农杆菌Agrobacterium介导侵染烟苗,检测其侵染效率,人工接种HCRV到沉默表达载体处理过的烟苗,通过观察发病症状、统计病情并应用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术检测TRV介导的基因沉默体系对HCRV侵染的沉默效应。结果表明,pTRV2-NSm沉默表达载体对本氏烟植株的侵染率达到95.00%,并且对本氏烟的生长无明显影响;与对照相比,经沉默载体处理的烟草植株接种HCRV后发病率显著降低,在接种后14 d发病率下降了90个百分点,病毒积累量明显下降,随着时间延长pTRV2-NSm对病毒的抑制效果持续升高,在接种后14 d对病毒病的防治效果最高,达93.13%;qRT-PCR检测发现基因沉默表达载体在21 d内能持续有效表达。表明本研究构建的基因沉默体系能有效沉默HCRV NSm的表达,同时降低HCRV在烟草上的发病率和积累量。In order to investigate the silencing effect of tobacco rattle virus(TRV)vector-mediated gene silencing technology on the movement protein gene NSm of Hippeastrum chlorotic ringspot virus(HCRV),a gene silencing expression vector pTRV2-NSm targeting the HCRV NSm gene sequence was constructed using tobacco(Nicotiana tabacum)as the material.The vector was transfected into tobacco plants through Agrobacterium mediated infection,and its infection efficiency was detected.HCRV was artificially inoculated into silencing vector-treated tobacco plants,and the symptoms were observed.The condition was statistically analyzed,and quantitative real-time polymerase chain reaction(qRTPCR)technology was applied to detect the silencing effect of TRV-mediated gene silencing systems on HCRV infection.The results showed that the infection rate of pTRV2-NSm silencing expression vector on the plant reached 95.00%,and there was no obvious effect on the normal growth of tobacco plants.Compared with the control,the incidence rate of tobacco plants inoculated with HCRV after treatment with silencing vector significantly decreased.The incidence rate dropped by 90 percentage points after 14 days,and the virus accumulation significantly decreased.With the extension of time,the inhibition effect of pTRV2-NSm on the HCRV infection continued to increase,and the inhibition effect was maximal at 14 days after inoculation,up to 93.13%.The qRT-PCR results revealed that gene silencing vectors were consistently and effectively expressed for 21 days.The gene silencing system constructed in this study can effectively silence the expression of HCRV NSm,while reducing the incidence and virus accumulation of HCRV in tobacco.

关 键 词：朱顶红褪绿环斑病毒 基因沉默 运动蛋白 病毒积累量 侵染效率 

分 类 号：S432.41[农业科学—植物病理学]