拟南芥钙调素结合蛋白cDNA克隆的分离和初步鉴定  

Isolation and Characterization of an Arabidopsis thaliana cDNA Encoding Calmodulin-binding Protein

作　　者：詹红利[1] 李翠风[1] 祁倩[1] 李国亮[1] 凌启阆[1] 

机构地区：[1]南开大学生物化学与分子生物学系,天津300071

出　　处：《南开大学学报（自然科学版）》2006年第3期6-11,共6页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：国家自然科学基金(30070385)

摘　　要：以生物素标记的钙调素为探针,从拟南芥(Arabidopsis thaliana)λZAPII cDNA表达文库中分离到9个阳性克隆．融合蛋白经CaM-gel overlay分析,其中Y1编码的融合蛋白在1 mmol/L Ca^(2+)存在下与胶体金标记的钙调素有明显结合．序列测定结果显示其外源片段全长为726 bp,含有一个476 bp的开放阅读框．GenBank数据库搜索及同源性分析结果表明该序列为拟南芥1号染色体基因组5661～7013序列,编码一未知蛋白,但与真核生物翻译起始因子5A(eIF-5A)具87%的同源性,并具相似的分子量、等电点和相同的保守序列．与eIF- 5A的结构特征比较,可初步确定Y1编码的蛋白为真核生物翻译起始因子5A家族中的新成员．即拟南芥1号染色体5661～7013序列为一尚未报道的翻译起始因子基因．蛋白质二级结构预测其C-端122～135氨基酸残基处有一双亲α-螺旋结构,这一结构与大多数钙调素结合蛋白中的钙调素结合位点的结构特征一致．该序列已提交NCBI BankIt数据库,登录号为AF492850．An Arabidopsis λZAPⅡ cDNA expression library was screend with biotinylated calmodulin as a probe, nine positive clones were isolated. The results of fusion protein＇s colloidal gold-calmodulin overlay indicated that the clone Y1 synthesized peptide that bound calmodulin in a Ca^2＋-dependent manner. Secondary structure prediction of Y1 protein showed that 122-135 residues formed an amphiphilic α-helix. This structure is in accordance with the structure characteristics of calmodulin binding domain, it is a probable calmodulin binding site. Sequencing showed that Y1 contained a 726 bp insert with an open reading frame of 476 bp, encoding 159 amino acid. Database search indicated that Y1 was genomic sequence 5 661- 7 013 of Arabidopsis chromasome No. 1 and encoded an unreported protein. Further study, revealed that Y1 had high sequence homology with eukaryotic translation initiation fac tot 5A（eIF-5A）. Besides, amino acid composition, secondary structure characteristics, molec ular wight and isoelectrotic point were similar to eIF-5A. Therefore, Y1 might be a new mem bet of elF 5A gene family. To our knowledge, none of the other eIF-5As so far are known to bind calmodulin, this is the first report to show that a potential elF 5A can bind calmodulin. The nucleotide sequence of Y1 has been reported to GenBank. Accession No. AF492850.

关 键 词：钙调素结构蛋白 真核生物翻译起始因子5A(eIF-5A) 表达文库筛选 

分 类 号：Q75[生物学—分子生物学]