棉花锌指蛋白GhZFP1相互作用蛋白的酵母双杂交筛选

Screening of Proteins Interacting with GhZFP1 Zinc Finger Protein from Cotton by Yeast Two Hybrid System

GhZFP1蛋白是从盐胁迫棉花幼苗cDNA文库中分离的一种CCCH型锌指蛋白.初步的生物学功能研究表明,过量表达该基因的转基因烟草耐盐性和抗病性显著提高.为深入研究GhZFP1蛋白的作用机制,构建pGBKT7-m1诱饵表达载体,利用酵母双杂交系统从盐胁迫诱导棉花cDNA文库中筛选与其相互作用的蛋白.通过阳性克隆的表型确定、PCR和限制性内切酶检测以及测序和生物信息学分析,获得9个与诱饵蛋白相互作用的靶蛋白.双分子荧光互补实验证明,GhZFP1与GZIRD19A确实存在互作关系.通过分析这些靶蛋白的已知功能,为研究GhZFP1锌指蛋白的未知生物学功能提供重要信息.

We isolated a novel CCCH-type zinc finger protein GhZFP1 （Gossypium hirsutum zinc finger protein 1） from a salt-induced cotton （Gossypium. hirsutum） cDNA library. It has been demonstrated that overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to disease. To get new clues and probe the unknown function and mechanism of GhZFP1,we isolated and identified proteins that interact with GhZFP1 by yeast two-hybrid system. The bait plasmids （ pGBKT7-GhZFP1,pGBKT7-m1,pGBKT7-m2 ） were constructed,respectively. By using the N-terminal 237 amino acids of GhZFP1 （m1） as a bait,we screened the salt-induced cotton leaves cDNA library. The positive clones were sequenced and further analyzed by bioinformatic approach. Nine positive clones interacting with GhZFP1 were identified. The interaction between GhZFP1 and GZIRD19A （ GhZFP1 interacting and responsive to dehydration protein 19A） was further confirmed by bimolecular fluorescence complementation （ BiFC ） visualization assay. Given the significant functional correlation between GhZFP1 and its interacting proteins,it will help to elucidate the possible mechanisms of GhZFP1 in improving the tolerance of transgenic plants.