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 Cu-cumber Mosaic Virus Banana Isolate
瞿青云 1沈文涛 1庹德财 2言普 2陈萍 3周鹏1
作者信息
- 1. 海南大学园艺学院,海口,570228;中国热带农业科学院热带生物技术研究所,海口,571101
- 2. 中国热带农业科学院热带生物技术研究所,海口,571101
- 3. 海南大学园艺学院,海口,570228
- 折叠
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-BanRNA1全长为3 375 bp,编码1a蛋白;CMV-Ban RNA2全长为3 043 bp,编码2a和2b蛋白;CMV-Ban RNA3全长为2 218 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YNRNA3同源性最高(94.5％).通过VectorNTI 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pCass4-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.
Abstract
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 mo-saic virus(CMV-Ban)was extracted from infected banana samples,and its triplet genome RNA was cloned and se-quenced.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 CMV-Ban 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 reached 99.9％.Phylogenetic tree analysis showed that the isolate CMV-Ban isolated from Hainan bananas infected with cu-cumber 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-Banl,RCCMV-Ban2,RC-CMV-Ban3 were successfully constructed,and Nicotiana benthamiana was infected by Agro-bacterium 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Key words
Musa spp./Cucumber mosaic virus/Evolutionary tree analysis/Invasive cloning引用本文复制引用
基金项目
中央级公益性科研院所基本科研业务费专项(19CXTD-33-1630052019015)
中央级公益性科研院所基本科研业务费专项(19CXTD-33-1630052019018)
出版年
2024
NETL
NSTL
维普
万方数据