黄瓜花叶病毒基因沉默载体的病毒含量和症状分析  

Analysis of Virus Content and Symptoms of Cucumber Mosaic Virus-based Gene Silencing Vector

作　　者：吴娟 李佳燕 李伟 曾紫怡 竺锡武 WU Juan;LI Jiayan;LI Wei;ZENG Ziyi;ZHU Xiwu(Institute of Agriculture and Biotechnology,Hunan University of Humanities,Science and Technology,Loudi Hunan 417000,China)

机构地区：[1]湖南人文科技学院农业与生物技术学院,湖南娄底417000

出　　处：《植物医学》2023年第6期21-27,共7页Plant Health and Medicine

基　　金：湖南省教育厅科研创新平台开放基金(18K100).

摘　　要：病毒诱导的基因沉默(Virus-induced Gene Silencing,VIGS)已被广泛应用于植物基因功能研究,具有操作容易、较短时间内即可观察到沉默效果、成本较低的特性.黄瓜花叶病毒(Cucumber Mosaic Virus,CMV)VIGS载体已经应用于沉默辣椒、烟草、大豆等植物基因,对于研究植物基因功能有重要作用.研究以CMV的Fny株系(CMV-Fny)RNA2中2b基因缺失载体CMV-F209△2b和表达2b基因N端61个氨基酸的载体CMV-F2092b61aa为对象,插入不同长度的外源基因gfp片段后,分析CMV VIGS载体在本氏烟植株上的病毒表达含量及诱导产生的病毒症状.结果表明,插入外源基因片段后,病毒外壳蛋白(Coat Protein,CP)基因RNA表达水平均明显降低.携带不同长度gfp序列的重组病毒CMVF2092b61aa-gfp 100、CMVF2092b61aa-gfp 200和CMVF2092b61aa-gfp 350其CP RNA含量比装载最适长度gfp序列的CMVF209△2b-gfp 350高10倍左右,且同样不引发明显病毒症状.由此可见,CMVF2092b61aa相比CMV-F209△2b更适合作为高效的VIGS载体,且初步探明它的最适插入片段长度为200~350 nt.Virus-induced gene silencing(VIGS)has been widely used for plant gene functional analysis.It is easy to operate,to observe the silencing effect in short time and low cost.Cucumber Mosaic Virus(CMV)has been successfully deployed as a VIGS vector in plants such as chili,tobacco and soybean,playing an important role in plant gene functional analysis.In this study,GFP gene fragments of different lengths were inserted into the 2b gene deletion vector CMV-F209△2b and CMV-F2092b61aa with truncated 2b to analyze the virus expression level and induced virus symptoms of these CMV VIGS vectors in Nicotiana benthamiana.The results indicated that the accumulation of CP RNA was significantly reduced after inserting foreign gene fragments.The CP RNA contents of recombinant viruses CMVF2092b61aa-gfp 100,CMVF2092b61aa-gfp 200,and CMVF2092b61aa-gfp 350 carrying different length of GFP sequences were about 10 times higher than that of CMVF209△2b-gfp 350 carrying the optimal length of GFP sequence,and they also did not cause obvious virus symptoms.Therefore,CMVF2092b61aa is more suitable as an efficient VIGS vector than CMV-F209△2b,and its optimal fragment length of insertion has been preliminarily determined to be 200~350 nt.

关 键 词：病毒诱导的基因沉默 黄瓜花叶病毒 2b 病毒含量 

分 类 号：S432[农业科学—植物病理学]