辣椒上TRV介导的番茄斑萎病毒N基因沉默体系构建及鉴定  

Establishment and Identification of TRV-mediated Tomato spotted wilt orthotospovirus N Gene Silencing System in Pepper

作　　者：李慧芳 桂敏 张萍萍 张美玲 李永忠[3] 刘雅婷[1] LI Huifang;GUI Min;ZHANG Pingping;ZHANG Meiling;LI Yongzhong;LIU Yating(College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;College of Plant Protection,Yunnan Agricultural University,Kunming 650201,China;College of Tobacco Science,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学农学与生物技术学院,云南昆明650201 [2]云南农业大学植物保护学院,云南昆明650201 [3]云南农业大学烟草学院,云南昆明650201

出　　处：《云南农业大学学报（自然科学版）》2021年第3期409-416,共8页Journal of Yunnan Agricultural University:Natural Science

基　　金：云南省重点项目(2018FA019);国家自然科学基金项目(31471828,31260451)。

摘　　要：【目的】研究番茄斑萎病毒(Tomato spotted wilt orthotospovirus,TSWV)核衣壳蛋白基因(N)对辣椒抗病性的影响。【方法】以易感TSWV的辣椒湘研11为研究对象,采用同源序列克隆获得辣椒CaPDS基因和TSWV N基因;利用烟草脆裂病毒(Tobacco rattle virus,TRV)和病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术构建辣椒CaPDS基因沉默载体(pTRV2-CaPDS)和TSWV N基因沉默载体(pTRV2-N),农杆菌GV3101注射接种辣椒,通过qRT-PCR检测重组载体沉默效率。【结果】湘研11接种含pTRV2-CaPDS载体菌液3周后,新叶出现白化现象,说明辣椒上的pTRV2-CaPDS沉默载体构建成功。qRT-PCR结果表明:pTRV2-N载体可高效沉默N基因,其表达量仅为6.79%±2.56%,说明沉默N基因后,湘研11不易感染TSWV。【结论】本研究成功构建了pTRV2-N沉默载体,沉默TSWV的N基因后辣椒对入侵的TSWV产生一定的抗性。[Purpose]To explore the impact of Tomato spotted wilt orthotospovirus(TSWV)nucleocapsid protein gene(N)on pepper disease resistance.[Method]Xiangyan 11,a pepper susceptible to TSWV,was used as the research object.Pepper phytoene desaturase(CaPDS)gene and TSWV N gene were cloned by homologous sequence.The CaPDS gene silencing vector(pTRV2-CaPDS)and TSWV N gene silencing vector(pTRV2-N)of pepper were constructed by using Tobacco rattle virus(TRV)and virus-induced gene silencing(VIGS)technology.Agrobacterium GV3101 was inoculated with pepper.The silencing efficiency of the recombinant vector was detected by qRT-PCR.[Result]Three weeks after inoculation with pTRV2-CaPDS,the new leaves appeared albino,which indicated that the construction of pTRV2-CaPDS silencing vector was successful.The qRTPCR results showed that the pTRV2-N vector can efficiently silence the N gene,and its expression is only 6.79%±2.56%,indicating that after silencing the N gene,Xiangyan 11 is not susceptible to TSWV.[Conclusion]pTRV2-N silencing vector was successfully constructed.After silencing the TSWV N gene,pepper had certain resistance to invading TSWV.

关 键 词：番茄斑萎病毒 病毒诱导的基因沉默 实时荧光定量PCR 烟草脆裂病毒 核衣壳蛋白基因 

分 类 号：S641.303.4[农业科学—蔬菜学]