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园艺学报 ›› 2008, Vol. 35 ›› Issue (7): 967-972.

• 果树 • 上一篇    下一篇

葡萄A病毒四川分离物的外壳蛋白基因克隆与原核表达

王建辉1,2;刘 晓2;席德慧1;袁 澍1;蒋 彧1;杨 辉1;杜俊波1;张中伟1;陈克玲2;林宏辉1*
  

  1. (1生物资源和生态环境教育部重点实验室,四川大学生命科学学院,成都 610064;2四川省农业科学院园艺研究所, 成都610066)
  • 收稿日期:2008-01-14 修回日期:2008-05-09 出版日期:2008-07-25 发布日期:2008-07-25
  • 通讯作者: 林宏辉

Cloning and Prokaryotic Expression of CP Gene of Grapevine Virus A Sichuan Isolate

WANG Jian-hui1,2, LIU Xiao2, XI De-hui1, YUAN Shu1, JIANG Yu1, YANG Hui1, DU Jun-bo1,ZHANG Zhong-wei1,CHEN Ke-ling2,and LIN Hong-hui1*
  

  1. [1Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610064, China; 2Horticulture Institute, Sichuan Academy of Agriculture and Science, Chengdu 610066, China]
  • Received:2008-01-14 Revised:2008-05-09 Online:2008-07-25 Published:2008-07-25
  • Contact: LIN Hong-hui

摘要: 葡萄A病毒(Grapevine Virus A,GVA)是葡萄病毒属(Vitivirus)的典型种,在世界葡萄产区广泛分布。采集10株“藤稔”葡萄成熟枝条,使用6种葡萄病毒ELISA试剂盒检测发现10个样本中有6个感染4种不同的葡萄病毒。以GVA的ELISA阳性植株为材料进行RT-PCR扩增,首次获得了GVA四川分离物SL10的完整外壳蛋白基因(CP)。该基因全长597 bp,将其与GeneBank收录的15个GVA分离物的CP序列进行比对和构建系统进化树。把不同地理起源的GVA分离物分成2个变异组;其中Ⅰ组包括3个分离物(与Ⅱ组的其他分离物只有75.9%~80.1%的序列同一性);其余的13个分离物组成Ⅱ组(组内分离物具有84.4%~99.5%的序列同一性)。构建了GVA CP的原核表达质粒PET-30-GVAcp并转化BL21菌株,经IPTG诱导,目的基因得到了大量表达。

关键词: 葡萄A病毒, ELISA, RT-PCR, 外壳蛋白基因, 原核表达

Abstract: Grapevine virus A (GVA) is a typical member in genus Vitivirus. The virus has been broadly discovered in vineyard around the world. Using an ELISA detection technology, four virus species were identified in 6 out of 10 shoot samples of Japanese hybrid grape, c.v. Fujiminori, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to amplify a complete coding sequence of the GVA coat protein (CP) from the virus-positive shoot samples. The CP gene, specified as the Sichuan isolate "SL10", contains 597 base pairs in length. The "SL10" CP gene was compared with other 15 GeneBank-collected CP genes, isolated from different geographic origins. Phylogenetic analysis clearly clustered the 16 CP genes into two distinguished groups. Group I contains only 3 members. The nucleotide identities among the 3 members range from 75.9% to 80.1%; whereas group II holds the rest 13 members. The nucleotide identities within the members in this group vary from 84.4% to 99.5%. The isolated CP gene was cloned into a prokaryotic expression vector and transformed into an E. coli strain BL21. After induced by IPTG, the CP gene was highly expressed in the E. coli.

Key words: Grapevine Virus A, ELISA, RT-PCR, coat protein gene, prokaryotic expression

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