摘要
钙调素结合蛋白的研究有助于探明钙调素介导的信号转导途径.以拟南芥钙调素亚型2(ACaM2)为钓饵,重组共转化法构建并筛选了酵母双杂交文库.复筛后得到一个阳性克隆.序列测定及分析表明,分离的阳性克隆中包含一个编码钙调素结合蛋白AtIQD26的cDNA片段.凝胶覆盖实验进一步表明,AtIQD26在1mmol/LCa2+或1mmol/LEGTA条件下都能与钙调素结合,说明其存在不依赖于Ca2+的CaM结合特性.GUS染色分析表明,AtIQD26具有普遍的组织表达特性,尤其是在新生的组织中表达量较大;融合荧光蛋白定位显示,AtIQD26在细胞核与质膜附近有分布.AtIQD26与钙调素空间分布的相似性,预示着它们在植物生长发育过程中可能共同发挥作用.
Calmodulin （CAM） is a ubiquitous, multifunctional calcium （Ca^2＋） sensor that exists in all eukaryotes. Calmodulin-binding proteins （CaMBPs） play important roles in various signal pathway of calmodulin. The finding of new CaMBPs will be useful for illustrating mechanism of CaM implicating in plant growth and development. Yeast two-hybrid system is an effective method for studying protein-protein interactions in vivo. In the study of plant signal transduction, many important signal transduction molecules were obtained by this system. Here, Arab idopsis flower were used as the material. The Yeast two-hybrid library of A rabidopsis flower was constructed by co-transforming yeast strain AH109 with ds cDNA, pGADT7-rec and pGBKT7-ACaM2. A positive clone was identified. DNA sequencing and analysis indicated that this cDNA clone encodes calmodulin-binding protein AtIQD26. The AtIQD26 protein contains a plant-specific domain of 67 conserved amino acid residues, referred to as the IQ67 domain（IQD）, which is characterized by a unique and repetitive arrangement of three different CaM recruitment motifs, known as the IQ, 1-5-10, and 1-8-14 motifs. Yeast two-hybrid analysis and gel overlay experiments demonstrated that AtIQD26 interacted with CaM both in the presence or absence of Ca^2＋. A striking feature of AtIQD26 is the high isoelectric point （-10.6） and frequency of serine residues （-10%）. To uncover potential roles for AtIQD26, a series of expression vectors were constructed about it, and the relative transgenic works were finished. Using these transgenic lines, the tissue expression and the subcellular localization of AtIQD26 were studied. Fusion GFP reporter showed that AtIQD26 was located at the nucleus and near cytoplasm membrane. GUS histochemical assay showed that AtIQD26 had characteristic of universal tissue expression, especially in the renascent tissue. Interaction of AtIQD26 with CaM and the presence of predicted CaM binding sites in it suggest that AtIQD26 is a new member of CaM targets. The basic isoelectric point and its potential nuclear localization suggest that AtIQD26 links Ca^2＋ signaling pathways to the regulation of gene expression. Expression similarity indicates that AtIQD26 combined with CaM to regulate plant development and growth.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2008年第6期703-711,共9页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金(2006CB0D1105,30470889,90208004)
河北省自然科学基金(C2004000152)
教育部新世纪优秀人才支持计划(NECT-06-0256)
河北师大博士基金资助项目(L2004B11).~~
关键词
钙调素结合蛋白
酵母双杂交
拟南芥
凝胶覆盖分析
组织表达
亚细胞定位
calmodulin-binding proteins, yeast two-hybrid system, Arabidopsis, gel overlay, tissue-specific expression, subcellular localization
