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作 者 ：王芳,杨金广,周本国,许大凤,王凤龙,高正良

期 刊 ：植物病理学报 2015年 45卷 6期 页码：569-575

关键词：马铃薯Y病毒；酵母双杂交；叶脉坏死症状；

Keywords:Potato virus Y, yeast two-hybrid system, vein necrosis symptom,

摘 要 ：马铃薯Y病毒坏死株系(Potato virus Y necrosis strain,PVY^N)侵染烟草后烟草表现叶脉坏死症状,但其致病的分子机制,特别是与病毒互作的宿主蛋白研究鲜有报道。PVY^N(JQ971975)的CKVNP编码区(CI-6K2-VPg-NIa-Pro区域,核苷酸5 540-6 491)与烟草表现叶脉坏死症状相关,研究其与寄主间的分子互作有助于揭示病毒侵染致烟草表现叶脉坏死症状的分子机制。本文将PVY^N的CKVNP编码区定向克隆到含有DNA结合功能域(DNA-binding Domain,BD)载体上,构建与激活功能域(Activation Domain,AD)融合表达的烟草cDNA文库,用CKVNP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CKVNP编码区可读框和氨基酸序列均正确,对酵母菌株无自主激活能力;烟草cDNA文库容量为7.0×10⁶ CFU/mL,且插入片段平均大小在1.0 kb以上,质量符合筛选要求;经过筛选和共转化回转验证,有94个候选基因与CKVNP在酵母中互作。挑选22个酵母质粒进行测序得到这些基因的cDNA序列,BLAST分析结果表明,这些基因共编码14种蛋白。

Abstract:Tobaccos infected by Potato virus Y N strain (PVY^N) usually exhibit vein necrosis symptom, but the mechanism has not been well understood, as no host proteins involved in the symptom has been reported yet. The previous studies have shown that polypeptide CKVNP (CI-6K2-VPg-NIa-Pro) coding region played an important role in inducing tobacco vein necrosis symptom. The bait plasmid (pGBK-PVY) containing the DNA-binding domain (BD) fused with full length of the polypeptide CKVNP encoded by PVY^N and the tobacco (Yunyan 87) cDNA library fused to the activation domain (AD) were constructed. Then tobacco cDNA library was screened by the CKVNP as a bait to identify protein(s) that interacted with CKVNP. The results showed that the amino acid sequence and open reading frame of the insert cDNA fragment of CKVNP were correct and it had no self activating effect on yeast strain. The cDNA library had a capacity of 7.0×10⁶ CFU/mL, and the most of insert fragments were more than 1 kb in size, which indicated that the cDNA library was qualified for the yeast two-hybrid screening. After screening and retesting the interaction in yeast 94 candidate genes displayed specific interaction with CKVNP in yeast. 22 genes were selected and sequenced. BLAST results showed that these genes encoded 14 proteins.

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