应用酵母双杂交系统筛选与CASTOR相互作用的蛋白.pdfVIP

农业生物技术学报 Journal ofAgricultural Biotechnology 2007，15 (6)： 1012～1018 · 研究论文· 应用酵母双杂交系统筛选与CASTOR相互作用的蛋白 杨 绮，朱 辉，陈春芬，朱 浩，张忠明 (华中农业大学农业微生物学国家重点实验室，武汉430070) 摘要：castor编码百脉根(Lotusjaponicus)中的离子通道蛋白，它是共生途径上的功能基因，作用于结瘤因子诱导产生的钙 离子激增(calcium spiking)信号转导途径的上游，突变后不能形成根瘤。为了进一步揭示castori~控共生信号途径的分子机制， 通过酵母双杂交系统在百脉根的eDNA文库中筛选可能与CASTOR相互作用的蛋白，以酵母(Saccharomyces cerevisiae)配合 的方法初筛获得 ll 1个阳性克隆，经X．Gal检测进一步确认有99个蓝色克隆子，测序以及NCBI blast比对分析鉴定出JAB1、 CLPA、钙调素结合的翻译延伸因子等7种可能与CASTOR相互作用的蛋白，利用RT．PCR方法检测了这些基因在接种以及 不接种根瘤菌(Mesorhizobium loti)的百脉根中的表达水平，分析推测CASTOR可能与上述蛋白形成复合物参与共生信号的转 导。 关键词：百脉根；CASTOR；酵母双杂交 中图分类号：S188 文献标识码：A 文章编号：1006．1304(2007)06．1012．07 Screening and Identification of Proteins Interacting with CASTOR by Yeast two—hybrid System YANG Qi，ZHU Hui，CHEN Chun—fen，ZHU Hao，ZHANG Zhong—ming (State KeyLaboratoryofAgriculturalMicrobiology,HuazhongAgricultural University,Wuhan 430070,China) A —瞅 The castor gene encodes predictable ion channel protein which is involved in nod factor signal transduction in Lotus japonicus．CASTOR mutants undergo root hairs deformation，but do not response to nod factors with intracellular calcium spiking．In order to investigate nod factor signal mechanisms，AD-eDNA library of L．japonicus was constructed．The 1 1 1 positive clones were identified using CASTOR as the bait at pre-screening by yeast two-hybrid approach．After retest the interaction in yeast (Saccharomyces cerevisiae)and X-Gal assay，99 positive clones were obtained．There were seven types of proteins including CaM binding translation elongation factor characterized by sequencing and NCBI blast analysis．By using RT-PCR，the expression level of these genes was further confirmed in L．japonicus with and without inoculation of Mesorhizobium loft，it was pr