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作 者 ：王祥；张秀春；余乃通；梁洁；周朋；刘志昕*

期 刊 ：植物研究 2015年 35卷 5期 页码：741-745

关键词：香蕉束顶病毒；nsp基因；酵母双杂交；诱饵载体；自激活；

Keywords:bananabunchytopvirus(BBTV), nspgenes, yeasttwo-hybrid, baitvector, self-activation,

摘 要 ：香蕉束顶病毒(Banana bunchy top virus,BBTV)DNA6编码的核穿梭蛋白(nuclear shuttle protein，NSP)在病毒的侵染、复制、运输中起重要作用。为了利用酵母双杂交系统研究BBTV DNA6与寄主香蕉蛋白的互作，本实验利用两对引物的PCR扩增得到BBTV -nsp片段，混合各自PCR产物进行熔化退火可得到1/4两端含有EcoRⅠ和BamHⅠ的酶切位点序列的DNA产物，将目的片段连接到酵母双杂交系统的pGBKT7诱饵载体中，成功获得pGBKT7-nsp，并将重组质粒pGBKT7-nsp转化入Y2H Gold酵母菌株中进行毒性检测和自激活验证。结果表明，pGBKT7-nsp没有自激活活性，同时对酵母细胞也无毒性，符合酵母双杂交诱饵质粒的要求，可用于下一步的蛋白互作实验。

Abstract:The nuclear shuttle protein(NSP) which encoded by banana bunchy top virus(BBTV) DNA6 plays an important role in infection, replication and transportation of virus. In order to research the interaction of BBTV DNA6 and host banana protein, the yeast two-hybrid system were used for the assay. pGBKT7-nsp genes was amplified using two pairs of specific primers, and their PCR products were mixed and amplified to obtain the 1/4 DNA products which contained EcoRⅠ and BamHⅠ enzyme site sequence. And then the DNA was cloned to pGBKT7 of yeast two-hybrid system. After confirmation with sequence analysis, the plasmid was transformed into the yeast cell Y2H, and its toxicity and transcriptional activation were tested by color assay. The pGBKT7-nsp had no selfactivation, and non-toxic to yeast cells, and it was consistent with the requirements of yeast two hybrid bait plasmids for the next step in the experiment.

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