《植物生理学报》 2009, 45(4): 372-378

细菌双杂交筛选大豆GmWNK1 互作蛋白系统的建立及应用

王应祥, 郑焱, 梁翠月, 王秀荣, 庄楚雄, 严小龙 , 廖红*

华南农业大学根系生物学研究中心, 广州510642

通信作者：廖红；E-mail: hliao@scau.edu.cn；Tel: 020-85283380

摘　要：

本研究利用大肠杆菌双杂交系统构建了一个高质量的大豆根系cDNA文库, 同时利用大肠杆菌双杂交表达载体pBT 构建了融合表达质粒pBT-GmWNK1, 经酶切和测序鉴定、诱饵融合蛋白的表达检测及诱饵融合蛋白的自激活鉴定后作为 诱饵, 从大豆根部 cDNA 文库中筛选与 GmWNK1 发生互作的蛋白质, 共获得 18 个阳性克隆。经测序和同源性比对发现, 有10 个阳性克隆编码已知蛋白, 8 个为假阳性。研究结果为揭示WNK基因家族的生物学功能和调控机制提供了重要的参 考数据和研究材料。

关键词：大豆; cDNA文库; 细菌双杂交; GmWNK1

收稿：2009-01-19   修定：2009-03-02

资助：国家科技部重大基础研究项目(2005CB120902)、国家自然科学基金(30230220)和中国博士后科学基金。

Establishment and Application of Bacterial Two-Hybrid System for Screening the Targets of GmWNK1 in Soybean

WANG Ying-Xiang, ZHENG Yan, LIANG Cui-Yue, WANG Xiu-Rong, ZHUANG Chu-Xiong, YAN Xiao-Long , LIAO Hong*

Root Biology Center, South China Agricultural University, Guangzhou 510642, China

Corresponding author: LIAO Hong; E-mail: hliao@scau.edu.cn; Tel: 020-85283380

Abstract:

A soybean root cDNA library was constructed using the bacterial two-hybrid system. A bait plasmid pBT-GmWNK1 was also constructed and sequenced. After expression detection and self-activation identification of pBT-GmWNK1 fusion protein in bacterial two-hybrid system, we screened GmWNK1 interacting proteins and isolated 18 positive clones. Among these 18 positive clones, 10 encoded known proteins and 8 were false positive. These results provided important data for us to further investigate the biological functions and regulation mechanism of with no lysine kinase (WNK) family.

Key words: soybean; cDNA library; bacterial two-hybridization system; GmWNK1