李属坏死环斑病毒(PNRSV)新疆巴旦木分离物外壳蛋白基因(CP)片断的克隆与序列分析  

Cloning and sequence analysis of CP gene of Prunus necrotic ringspot virus from almond plants in Xinjiang

作　　者：殷智婷[1] 韩剑[1] 周国辉[2] 张祥林 罗明[1] 潘亚南[1] 

机构地区：[1]新疆农业大学农学院,乌鲁木齐830052 [2]华南农业大学资源与环境学院,广州510642 [3]新疆出入境检疫检验局,乌鲁木齐830052

出　　处：《果树学报》2012年第5期740-746,共7页Journal of Fruit Science

基　　金：新疆自治区自然科学基金(2012211B25);新疆自治区科技支疆项目(200991238)

摘　　要：【目的】为了查明新疆巴旦木果树病原病毒的种类,为病毒的分子检测提供基础。【方法】采用双抗体夹心酶联免疫吸附法(DAS-ELISA),从巴旦木果树叶片中检测到李属坏死环斑病毒(PNRSV)。以阳性样品的总RNA为模板进行PNRSV外壳蛋白基因(CP)RT-PCR扩增,对预期430 bp大小的4个扩增产物进行克隆、测序和序列分析。【结果】结果表明,PNRSV新疆巴旦木分离物CP基因的核苷酸和氨基酸序列与世界各地已报道的分离物具有较高的同源性,分别为80.0%～97.2%和80.1%～94.1%,其中与阿根廷分离物AY217的同源性最高;而与同属03亚组的苹果花叶病毒(ApMV)同源性较低,仅为52.9%～55.4%和45.6%～48.6%。新疆PNRSV各分离物之间CP基因高度同源。序列比对与系统发生树的分析显示,PNRSV新疆巴旦木分离物的株系属于GroupⅠ组。【结论】确定了PNRSV为侵染新疆巴旦木果树的病原病毒。这是PNRSV在新疆发现的首次报道。[Objective]The objective of the study is to identify the virus infecting almond trees in Xinjiang Province,and provide a foundation for developing molecular detection of the viral diseases. [Method]The samples showing typical symptoms of virus-like diseases were screened by Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assays （DAS-ELISA）, and eight were tested positive for Prunus necrotic ringspot virus （PNRSV）. Using universal primers for viral coat protein gene （CP） of PNRSV, RT-PCR were performed with the total RNA as templates extracted from the detected positive samples. Four amplified cDNA fragments with expected amplified sizes of 430 bp were cloned and sequenced. [Result]The similarity analysis of the nucleotide sequences and deduced amino acid of CP gene showed highly identity between of PNRSV-Xinjiang isolates and that from several different countries, sharing identities of 80.0% to 97.2% and 80.1% to 94.1% respectively. They shared the highest identity with PNRSV-Argentina isolates AY217, while less than 52.9% to 55.4% and 45.6% to 48.6% when compared with Apple mosaic virus （ApMV） which belong to the same subgroup of Ilarvirus. High sequence identities of CP gene were showed among Xinjiang isolates. Xinjiang isolates were clustered to Group I （the serious pathotype） in phylogenetic analysis. [Conclusion]It was concluded that PNRSV infected almond in Xinjiang. This is the first report that PNRSV was detected in Xinjiang, China.

关 键 词：巴旦木 李属坏死环斑病毒(PNRSV) 双抗体夹心酶联免疫吸附法(DAS—ELISA) RT—PCR CP基因 

分 类 号：S662.9[农业科学—果树学]