木薯普通花叶病毒侵染性克隆及GFP表达载体的构建与分析  

Infectious Clone of Cassava Common Mosaic Virus and Construction and Analysis of GFP Expression Vector

作　　者：俞春烨 庹德财[2] 沈文涛[2] 言普[2] 黎小瑛[2] 周鹏 徐晶宇[1] Yu Chunye;Tuo Decai;Shen Wentao;Yan Pu;Li Xiaoying;Zhou Peng;Xu Jingyu(College of Agricultural,Heilongjiang Bayi Agricultural University,Daqing,163319;Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Science,Haikou,571101)

机构地区：[1]黑龙江八一农垦大学农学院,大庆163319 [2]中国热带农业科学院热带生物技术研究所,海口571101

出　　处：《分子植物育种》2022年第18期6030-6040,共11页Molecular Plant Breeding

基　　金：国家自然科学基金青年科学基金项目(32000399)资助。

摘　　要：木薯普通花叶病毒(Cassava common mosaic virus,CsCMV)(Potexvirus属,Alphaflexiviridae科)是新发现的一种潜在威胁木薯产业的病毒。本研究以前期在中国首次报道发现的CsCMV海南儋州Hainan-DZ分离物(CsCMV-HN)为基础,利用In-Fusion无缝克隆将CsCMV-HN全长cDNA片段克隆到植物表达载体pGreenII-35S上,构建其全长cDNA侵染性克隆pCsCMV-HN。通过农杆菌介导法将侵染性克隆pCsCMV-HN注射接种本生烟,接种10 d后开始出现褪绿及叶皱缩等症状,经RT-PCR检测和病症观察统计,侵染效率达100%。在此基础上,采用90 bp重复的CP亚基因组启动子(subgenomic RNA promoter,SGP)策略构建其携带gfp报告基因的病毒表达载体pCsCMV-GFP,农杆菌注射接种本生烟7~10 d后,在紫外灯下可观察到非接种叶有较强的绿色荧光,RT-PCR检测和Western blot分析表明pCsCMV-GFP可在本生烟上系统性表达GFP。本研究成功构建的CsCMV侵染性克隆及GFP表达载体将为进一步研究该病毒基因功能及致病机制提供借鉴。Cassava common mosaic virus(CsCMV)(genus Potexvirus,family Alphaflexiviridae)is an emerging threat to cassava production in China.Here,an agroinfection-compatible infectious CsCMV cDNA clone designated pCsCMV-HN were successfully constructed using the one-step In-Fusion Cloning based on the complete genome sequence of Cs CMV Hainan-DZ islolate(CsCMV-HN),which was first reported in China.The Nicotiana benthamiana plants agroinoculated with agrobacterium-carrying pCsCMV-HN exhibited mosaic and chlorosis symptoms at 10 days post inoculation(dpi).The infection efficiency reached 100%by RT-PCR and disease observation.On this basis,the viral expression vector PCSCMV-GFP carrying the gfp reporter gene was constructed by using the90 bp repeat CP subgenomic RNA promoter(SGP)strategy.At 7~10 days after inoculation with agroinfection,strong green fluorescence was observed in the non-inoculated leaves under UV light.RT-PCR and Western blot analysis showed that p CsCMV-GFP could systematically express GFP on N.benthamiana.The successful construction of Cs CMV infection clone and GFP expression vector in this study will provide reference for further study of the gene function and pathogenic mechanism of CsCMV.

关 键 词：木薯普通花叶病毒 侵染性克隆 植物病毒表达载体 绿色荧光蛋白 本生烟 

分 类 号：S435.33[农业科学—农业昆虫与害虫防治]