葡萄A病毒四川分离物的外壳蛋白基因克隆与原核表达

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

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597bp。将其与GenBank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分离物分成两个变异组，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GVACP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。

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, cv. 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 ＂SLIO＂, contains 597 base pairs in length. The ＂SLIO＂ CP gene was compared with other 15 GenBank-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. coll.