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期 刊 ：植物保护学报 2010年

关键词：cDNA文库；酵母双杂交筛选；西瓜；诱饵质粒；

Keywords:cDNA library, yeast two-hybrid screening, Citrullus lanatus, bait plasmid,

摘 要 ：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2.94×10⁶ CFU/mL,且大多数插入片段在700bp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。

Abstract:Movement protein (MP) is encoded by plant virus meditates viral movement in plant. Study on molecular interactions between MP and host is essential to elucidate the molecular mechanisms underlying the viral infection process. In this study, the bait plasmid (pGBK-CA_MP) with the full length of the MP encoded by Cucurbit aphid-borne yellows virus (CABYV) fused to the DNA-binding domain (BD), watermelon (Citrullus lanatus) cDNA library fused to the activation domain (AD) were constructed, and then watermelon cDNA library was screened by the MP as a bait to identify protein(s) that interacted with MP. The results showed that the amino acid sequence and open reading frame of the insert cDNA fragment of MP were correct, and it had no self-activating effect on yeast strain AH109 and Y187. The cDNA library had a titer of 2.94×10⁶ CFU/mL, and the most of insert fragments were more than 700 bp in size, which indicated that the cDNA library was qualified for the yeast two-hybrid screening. After screening and retesting the interaction in yeast, 48 candidate positive clones displayed specific interaction with MP in yeast and cDNA sequences were obtained. BLAST results showed that these cDNAs enco- ded 12 proteins.

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