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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 CMV-BHI外壳蛋白基因在香蕉中表达打下了基础
Banana leaf mosaic virus was infected from Daejeon in Hainan Island to obtain banana mosaic virus and its RNA was purified. The first strand of c DNA was synthesized by AMV reverse transcriptase, and a DNA of about 70 bp was obtained by PCR The results showed that the cloned DNA fragment contained a complete CMV-BHI coat protein gene with a length of 657 bp. The DNA fragment was then cloned into pBI1 2 1 and p KHG4 plasmid to construct two plant expression vectors containing the CaMV 35s promoter (5’-end), the NOS terminator (3’-end), and the NPT marker gene and the NPT and HPT marker genes, respectively (pTBB and pTTK ). Then, the pBIAH promoter was replaced with the UBI promoter in pAHC18 to construct pBIAH. The CMV-BHI coat protein gene was used to replace the GUS gene in pBIAH to construct a monocotyledonous plant promoter UBI And NPT marker gene plant expression vector (p TBBU). Thus laying a foundation for the expression of the CMV-BHI coat protein gene in banana