柑橘碎叶病毒外壳蛋白基因的克隆和序列分析  

Cloning and Sequence Analysis of the CP Gene of Citrus tatter leaf virus

作　　者：宋震[1] 刘科宏[1] 杨方云[1] 唐科志[1] 李中安[1] 周常勇[1] 

机构地区：[1]西南大学柑橘研究所/中国农业科学院柑桔研究所,重庆400712

出　　处：《中国农业科学》2009年第10期3741-3748,共8页Scientia Agricultura Sinica

基　　金：重庆市科技攻关项目(CSTC;2007AA1024);国家科技支撑计划(2007BAD61B04)

摘　　要：【目的】了解中国CTLV分离物外壳蛋白(CP)基因的变异情况。【方法】对来源于中国不同地区、不同寄主品种的18个柑橘碎叶病毒(CTLV)分离物进行RT-PCR、克隆、测序,应用DNAMAN软件进行序列分析。【结果】18个分离物的CP基因全长714nt,推导的CP含237个氨基酸。CP核苷酸序列及推导的氨基酸序列最大相似性分别为88.5%～99.9%和91.1%～99.6%。与核苷酸序列G289→A或C、A409→C和G414→T的单碱基突变相对应,CTLV外壳蛋白第97、137、138位氨基酸在强、弱毒分离物之间存在差异,多数弱毒分离物为Q97或K97、Q137、H138,而多数强毒分离物为E97、R137或K137、Q138。在根据CP氨基酸序列构建的系统进化树上,本研究获得的18个CTLV分离物至少可以划分为2个组群,其中,在指示植物上表现较弱症状的多数(4/6)CTLV分离物划分在第Ⅰ组群,多数(10/12)CTLV强毒分离物划分在第Ⅱ组群。【结论】CTLV外壳蛋白基因相对保守,其第289、409、414位碱基在强、弱毒分离物之间存在差异,可能与病毒致病性相关。【Objective】 This study was conducted to investigate the molecular variability of the coat protein （CP） gene of Citrus tatter leaf virus （CTLV） isolates collected from China. 【Method】 The CP gene of 18 CTLV isolates from different geographical origins and citrus varieties were amplified by reverse transcription-polymerase chain reaction （RT-PCR）. The DNA products were cloned and sequenced,and sequence analysis was conducted by DNAMAN. 【Result】 The CP gene of 18 CTLV isolates were all 714 nucleotides in length,and the putative CP contained 237 amino acids （aa）. The identities of nucleotide and deduced amino acid sequences of CP gene among 18 isolates ranged from 88.5% to 99.9% and 91.1% to 99.6%,respectively. Corresponding to the mutation G^289→A or C,A^409→C,and G^414→T on nucleotide sequences of CP gene,amino acids at positions 97,137,and 138 of CP were different between isolates expressing mild symptoms and isolates expressing severe symptoms in the indicators. Q^97 or K^97,Q^137,H^138 were discovered in most mild isolates,whereas E^97,R^137 or K^137,Q^138 were shared by most severe isolates. Phylogenetic trees based on the aa sequences of the coat proteins showed that 18 CTLV isolates were divided into two clusters,and most of the mild isolates （4/6） belonged to the Ⅰg roup and most severe isolates （10/12） belonged to the Ⅰgroup. 【Conclusion】 The CP gene of CTLV was relatively conserved. The differences between mild and severe strains are very subtle,with three nucleotide positions （289,409 and 414） appearing to determine the pathogenicity of virus.

关 键 词：柑橘碎叶病毒 CTLV 外壳蛋白基因 克隆 序列分析 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]