香蕉花叶病毒外壳蛋白基因克隆及表达载体的构建

Cloning and reconstruction of expression vector of the banana mosaic virus coat protein gene

从海南大田感染香蕉花叶病的香蕉叶片 ,获得香蕉花叶病毒 ,提纯其 RNA,在 AMV反转录酶作用下合成 c DNA第一链 ,经 PCR扩增 ,获得一约 70 0 bp的 DNA片段 ,测序结果显示所克隆的 DNA片段包含一完整的香蕉花叶病毒株系 ( CMV-BHI)外壳蛋白基因 ,长度为 6 5 7bp,然后将此 DNA片段 ,分别克隆到p BI1 2 1和 p KHG4质粒 ,构成两个含 Ca MV35 s启动子 ( 5 '-端 )、NOS终止子 ( 3'-端 )和分别含 NPT 标记基因和 NPT 及 HPT标记基因的植物表达载体 ( p TBB和 p TBK)。然后用 p AHC1 8中的 UBI promoter换下p BI1 2 1的 Ca MV35 s promoter,构成 p BIAH;再用 CMV-BHI外壳蛋白基因换下 p BIAH中 GUS基因 ,构成一含单子叶植物启动子 UBI和 NPT 标记基因的植物表达载体 ( p TBBU)。从而为

A DNA fragment about 700 bp obtained from the first strand of cDNA which synthesized with a viral RNA template extracted from viral particles (CMV-BHI) isolated from propagation host banana in Hainan by using AMV reverse transcriptase and 3'-end PCR primer after 35 PCR amplification cycles. It was cloned into T-Easy vector. The whole DNA sequence was determined and the results showed that the entire gene (657 bp) encoding the coat protein had been cloned. Then two plant expression vectors (pTBB and pTBK) were reconstructed with the coat protein gene being cloned into pBI121 and pKHG4 respectively. And the third plant expression vector (pTBBU) was constituted by UBI promoter digested from pAHC18 exchanging with CaMV 35s promoter of pBI121, and then using 657 bp coat protein gene exchanging the GUS gene. All the three recombinants were screened with endonuelease digesting and DNA dot blot. This found the expression of CMV-BHI coat protein gene in banana plant.