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植物病毒编码的移动蛋白(movement protein,MP)介导病毒在寄主体内的移动,研究其与寄主间的分子互作有助于揭示病毒侵染过程中的分子机制。将南瓜蚜传黄化病毒Cucurbit aphid-borne yellows virus(CABYV)MP基因定向克隆到含有DNA结合功能域(DNA-binding domain,BD)载体上,并构建与激活功能域(activation domain,AD)融合表达的西瓜茎叶cDNA文库,然后用MP为诱饵筛选文库寻找与其互作的寄主因子。结果表明,诱饵质粒插入的MP基因可读框和氨基酸序列均正确,对酵母菌株AH109和Y187没有自主激活能力;文库滴度为2.94×106CFU/mL,且大多数插入片段在700bp以上,质量符合筛选要求;经过筛选和共转化回转验证,有48个候选阳性克隆与MP在酵母中互作。测序得到这些克隆的cDNA序列,BLAST分析结果表明,这些克隆共编码12种蛋白。
The movement protein (MP) encoded by plant virus mediates the movement of the virus in the host and its molecular interaction with the host is helpful to reveal the molecular mechanism of the virus infection. The gene of Cucurbit aphid-borne yellows virus (CABYV) MP was cloned into a DNA-binding domain (BD) vector and fused with activation domain (AD) Expression of stem and leaf cDNA library of watermelon, and then use MP as bait screening library to find the host factor interacting with it. The results showed that both the open reading frame and the amino acid sequence of the MP gene inserted into the bait plasmid were correct, and no self-activation ability to the yeast strains AH109 and Y187. The library titer was 2.94 × 106 CFU / mL and most of the inserted fragments were above 700 bp, Screening requirements; After screening and co-transformation rotation validation, 48 candidate positive clones interacted with MP in yeast. The cDNA sequences of these clones were sequenced. BLAST analysis showed that these clones encode 12 proteins.