拟南芥半胱氨酸蛋白酶抑制剂基因AtCYS6克隆及功能分析

Molecular Cloning and Functional Analysis of Cysteine Protease Inhibitor Gene AtCYS6 in Arabidopsis thaliana

为了研究拟南芥半胱氨酸蛋白酶抑制剂AtCYS6基因在植物生长发育过程中的作用与功能,从拟南芥中克隆了半胱氨酸蛋白酶抑制剂AtCYS6基因,并对该基因进行生物信息学分析,亚细胞定位分析,蛋白质诱导、表达、纯化及功能研究。结果表明,AtCYS6蛋白等电点为5.85,化学式为C1179H1848N322O348S6,共含有3703个原子数,为亲水性蛋白;AtCYS6与十字花科的荠菜、荠蓝的同源性较高,同源性分别为81.1%,80.3%,同水稻、玉米和大豆的同源性较低,同源性分别为55.5%,54.3%,56.6%,且在氨基酸序列中间具有高度保守的Q-V-G(Gln-Val-Gly)序列;同时进化树分析显示AtCYS6与同属于十字花科的荠菜、荠蓝的亲缘性较近,同水稻、玉米、高粱、谷子等禾本科植物的亲缘性较远;亚细胞定位结果显示其定位于细胞质,与预测结果相符合;蛋白质诱导、表达和纯化得到大小约为52 ku的融合蛋白,其中AtCYS6蛋白质的分子量约为26 ku,与预测值相符合;Western Blot分析显示,诱导表达的融合蛋白正确翻译表达,且没有出现结构上的断裂或者降解的情况;蛋白酶活性分析表明,AtCYS6对木瓜蛋白酶具有抑制作用。研究结果可以为下一步在植物体内研究该基因的相关机理作用提供基础。

In order to study the role and function of the cysteine protease inhibitor AtCYS6 gene in plant growth and development,the AtCYS6 gene was cloned from Arabidopsis thaliana.Bioinformatics analysis,subcellular localization analysis,protein induction,expression,purification and functional were carried out to study this gene.The results showed that the isoelectric point of AtCYS6 protein was 5.85,the chemical formula was C1179H1848N322O348S6,which contained 3703 atoms and it was a hydrophilic protein;AtCYS6 had high homology with Capsella rubella and Camelina sativa,the homology was 81.1%and 80.3%respectively,and it had low homology with Oryza sativa,Zea mays and Glycine max,the homology was 55.5%,54.3%and 56.6%respectively,among the amino acid sequences,there was a highly conserved Q-V-G(Gln-Val-Gly)sequence;evolutionary tree analysis showed that AtCYS6 was closely related to Capsella rubella and Camelina sativa,but far from Oryza sativa,Zea mays,Sorghum bicolor and Setaria italic;subcellular localization results showed that it was located in cytoplasm,which was consistent with the predicted results;the fusion protein was induced,expressed and purified to be about 52 ku in size,the molecular weight of AtCYS6 protein was about 26 ku,which accorded with the predicted value;Western Blot analysis showed that the fusion protein was correctly translated and expressed without structural breakage or degradation;enzyme activity analysis showed that AtCYS6 had inhibitory effect on papain.The results of this study provide a basis for further study of the related mechanism of AtCYS6 in plants.