黄瓜花叶病毒香蕉分离物基因组全长克隆及侵染性克隆构建  

The Genome Full-length Cloning and Invasive Cloning Construction of Cucumber Mosaic Virus Banana Isolate

作　　者：瞿青云 沈文涛 庹德财[2] 言普[2] 陈萍[1] 周鹏[1,2] Qu Qingyun;Shen Wentao;Tuo Decai;Yan Pu;Chen Ping;Zhou Peng(School of Horticulture,Hainan University,Haikou,570228;Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Science,Haikou,571101)

机构地区：[1]海南大学园艺学院,海口570228 [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》2024年第1期97-107,共11页Molecular Plant Breeding

基　　金：中央级公益性科研院所基本科研业务费专项(19CXTD-33-1630052019015,19CXTD-33-1630052019018)资助。

摘　　要：香蕉(Musa spp.)是重要的热带经济作物。为了解黄瓜花叶病毒(cucumber mosaic virus,CMV)对香蕉的侵染机制进而有针对性地进行防控,本研究从染病的香蕉样本中提取得到黄瓜花叶病毒(CMV-Ban),并对其三分体基因组RNA进行全长克隆和序列分析。其中,CMV-Ban RNA1全长为3375 bp,编码1a蛋白;CMV-Ban RNA2全长为3043 bp,编码2a和2b蛋白;CMV-Ban RNA3全长为2218 bp,编码3a运动蛋白和外壳(CP)蛋白。通过BLAST对比,CMV-Ban RNA1的核苷酸序列与马来西亚黄瓜分离株CLW2 RNA1的同源性最高(92.1%),CMV-Ban RNA2的核苷酸序列与意大利辣椒分离株(Vir)的同源性最高(91.8%),CMV-Ban RNA3与国内报道的油菜分离株YN RNA3同源性最高(94.5%)。通过Vector NTI Advance 11.5对比,CMV-Ban与台湾香蕉分离株20氨基酸序列同源性可达99.9%。通过进化树分析可知,从感染了黄瓜花叶病毒的海南香蕉上分离出来的分离物CMV-Ban属于亚组IB。采用In-Fushion无缝克隆将CMV-Ban三分体基因组RNA分别插入植物表达载体p Cass4-RZ,成功构建侵染性载体RC-CMV-Ban1、RC-CMV-Ban2、RC-CMV-Ban3,并通过农杆菌渗透注射技术侵染本生烟(Nicotiana benthamiana),21 d后叶片呈现明显的花叶斑驳症状,说明侵染性克隆RC-CMV-Ban成功侵染本生烟。研究结果为后续解析CMV-Ban对香蕉的侵染机制与防治提供科学依据。Bananas(Musa spp.),an important tropical cash crop.In order to understand the mechanism of cucumber mosaic virus(CMV) infection of bananas and then to prevent and control them in a targeted manner,cucumber mosaic virus(CMV-Ban) was extracted from infected banana samples,and its triplet genome RNA was cloned and sequenced.Among them,cmV-Ban RNA1 has a total length of 3 375 bp,encoding the 1a protein;CMV-Ban RNA2 is 3 043 bp in total length and encodes 2a and 2b proteins;CMV-Ban RNA3 is 2 218 bp in total length and encodes 3a kinematic protein and shell(CP) protein.By BLAST comparison,the nucleotide sequence of CMV-Ban RNA1 had the highest homology with CLW2 RNA1 of the Malaysian cucumber isolate(92.1%),the nucleotide sequence of CMVBan RNA2 had the highest homology with the Italian pepper isolate(Vir)(91.8%),and the homology of CMV-Ban RNA3 had the highest homology with the domestically reported rapeseed isolate YN RNA3(94.5%).Compared with Vector NTI Advance 11.5,the 20 amino acid sequence homology of CMV-Ban and Taiwan banana isolates reached99.9%.Phylogenetic tree analysis showed that the isolate CMV-Ban isolated from Hainan bananas infected with cucumber mosaic virus belonged to subgroup IB.After that,the CMV-Ban tripenomy genomic RNA was inserted into the plant expression vector pCass4-RZ using In-Fushion seamless cloning,and the invasive vectors RC-CMV-Ban1,RCCMV-Ban2,RC-CMV-Ban3 were successfully constructed,and Nicotiana benthamiana was infected by Agrobacterium osmotic injection technology,and the leaves showed obvious mosaic symptoms after 21 days,indicating that the infected clone RC-CMV-Ban successfully infected Nicotiana benthamiana.The results provide a scientific basis for the subsequent analysis of the infection mechanism and control of banans by CMV-Ban.

关 键 词：香蕉 黄瓜花叶病毒 进化树分析 侵染性克隆 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治]