《植物生理学报》 2014, 50(6): 772-778

AtSARK互作蛋白的筛选与鉴定

肖冬, 徐欣欣, 管婧雯, 王宁宁*

南开大学生命科学学院, 天津300071

通信作者：王宁宁；E-mail: wangnn@nankai.edu.cn；Tel: 022-23504096

摘　要：

植物LRR型类受体蛋白激酶在植物生命活动中发挥着重要作用。前期研究发现了一个通过生长素和乙烯的协同作用参与拟南芥叶片衰老过程正调控的类受体蛋白激酶, AtSARK。为深入研究 AtSARK 蛋白的作用机制, 文章采用酵母双杂交系统, 以AtSARK胞内域(AtSARK-CD)为诱饵蛋白, 对拟南芥均一化cDNA文库进行筛选, 得到83个AtSARK的候选互作组分, 经过复筛, 初步发现AtSARK的互作组分包括14-3-3蛋白、FKBP-型肽脯氨酰顺反异构酶、醛缩酶超家族成员、RING/U-box 超家族成员以及WRKY转录因子家族成员。我们对其中一个功能未知的醛缩酶家族基因AtFBA5 (AT4G26530)的表达模式进行了分析, 并用GST pulldown实验证实了AtSARK-CD与 AtFBA5间存在直接的相互作用。At-SARK互作组分的文库筛选及AtFBA5的分离有利于进一步研究 AtSARK 基因在衰老信号通路中的作用机制。

关键词：AtSARK; 拟南芥; 酵母双杂交; pulldown

收稿：2014-01-22   修定：2014-03-27

资助：国家自然科学基金项目(31170261)、教育部科学技术研究重大项目(313032)和教育部博士点基金优先发展领域项目(20130031130003)。

Identification of the Interactive Proteins of AtSARK

XIAO Dong, XU Xin-Xin, GUAN Jing-Wen, WANG Ning-Ning*

College of Life Sciences, Nankai University, Tianjin 300071, China

Corresponding author: WANG Ning-Ning; E-mail: wangnn@nankai.edu.cn; Tel: 022-23504096

Abstract:

Lucine-rich-repeat receptor-like kinases (LRR-RLKs) are known to function in various signaling pathways in plants. Our previous study suggested a role for an Arabidopsis LRR-RLK, AtSARK, which regulates leaf senescence through synergistic actions of auxin and ethylene. This study was designed to identify the interaction partners of AtSARK. A normalized Arabidopsis (Arabidopsis thaliana) cDNA library was screened by the yeast two hybrid screening method with the cytoplasmic domain of AtSARK (AtSARK-CD) as bait, and 83 candidate preys were identified. We confirmed five proteins (14-3-3 protein, FKBP peptidyl-prolyl cis-trans isomerase family protein, Aldolase superfamily protein, RING/U-box superfamily protein and a member of the plant WRKY transcription factor family) as the AtSARK interaction partners in yeast. Furthermore, we analyzed the expression pattern of AtFBA5 (AT4G26530), a function unknown gene of Aldolase superfamily, and confirmed the physical interaction between AtSARK-CD and AtFBA5 by an in vitro pulldown assay. The separation of AtSARK-interacting proteins is beneficial to disclose the mechanism of AtSARK in leaf senescence process.

Key words: AtSARK; Arabidopsis thaliana; yeast two hybrid system; pulldown