和TaFRA蛋白相互作用候选蛋白的筛选.pdfVIP

HEREDITAS (Beijing) 2011 1 , 33(1): 88―94 ISSN 0253-9772 研究报告 DOI: 10.3724/SP.J.1005.2011.00088 与TaFRA蛋白相互作用候选蛋白的筛选 温小杰, 郝晨阳, 蒲文, 刘旭, 张学勇 , , , 100081 F-box 蛋白是E3 泛素连接酶SCF 复合体的重要亚基, 通过底物蛋白的特异识别发挥功能。TaFRA 是在盐 胁迫差异表达片段基础上通过RACE 方法获得的一个基因, 编码F-box 蛋白。文章利用TaFRA 基因构建诱饵表 达载体, 直接用 cDNA+pGAD+pBD 共转化酵母双杂交的方法筛选相互作用的候选蛋白。通过对阳性克隆的鉴 定和测序分析, 共获得44 个与TaFRA 相互作用的候选蛋白, 其中32 个为已知蛋白, 包括硫氧还蛋白、金属硫 蛋白、ATP 合成酶及丝氨酸/ 苏氨酸蛋白激酶等多种逆境胁迫反应蛋白及转录因子蛋白, 说明 TaFRA 与胁迫反 应相关, 可能通过对上述蛋白编码基因的调控参与了植物的胁迫反应过程, 为进一步阐明TaFRA 的功能及作用 机制奠定了理论基础。 酵母双杂交; 共转化; TaFRA; 相互作用; 候选蛋白 Screening of the candidate proteins interacting with TaFRA WEN Xiao-Jie, HAO Chen-Yang, PU Wen, LIU Xu, ZHANG Xue-Yong The National Key Facility for Crop Gene Resources and Genetic Improvement, Key Laboratory of Crop Germplasm and Utilization, Min- istry of Agriculture, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China Abstract: F-box protein is an important subunit of SCF complex, an E3 ligase in ubiquitin system, and its function is determined through mediating the specific recognition and combining with substrate protein. TaFRA (F-box protein related to abiotic stress) was identified by RACE based on the fragments diferently expressing in wheat seedling exposed to salt stress and encodes an F-box protein. In this study, pBD-TaFRA bait expression vector was constructed, and cDNA+pGAD+pBD was directly co-transformed into yeast hybrid system to screen condidate proteins interacting with TaFRA. Fourty-four candidate proteins were obtained, in which 32 were known proteins and transcript factors related to stress toleranc