甘蓝型油菜γ-谷氨酰半胱氨酸合成酶(GCS)基因的克隆及生物信息分析  

Cloning and Bioinformatics Analysis ofγ-GCS Gene from Rapeseed(Brassica napus L.)

作　　者：贺晓岚 王建伟 陈新宏[3] 李文旭[4] 秦绍钊 He Xiaolan;Wang Jianwei;Chen Xinhong;Li Wenxu;Qin Shaozhao(School of Life and Health Science,Kaili University,Kaili,556011;School of Science,Kaili University,Kaili,556011;Shaanxi Key Laboratory of Genetic Engineering for Plant Breeding,College of Agronomy,Northwest A&F University,Yangling,712100;Institute for Wheat Research,Henan Academy of Agricultural Sciences,Zhengzhou,450002)

机构地区：[1]凯里学院大健康学院,凯里556011 [2]凯里学院理学院,凯里556011 [3]西北农林科技大学农学院,陕西省植物遗传工程育种重点实验室,杨凌712100 [4]河南省农业科学院小麦研究所,郑州450002

出　　处：《分子植物育种》2023年第2期379-388,共10页Molecular Plant Breeding

基　　金：贵州省教育厅科技拔尖人才支持计划项目([2017]094);贵州省科技支撑计划项目([2017]2522);贵州省科技计划项目([2017]1167)共同资助。

摘　　要：谷胱甘肽(glutathione,GSH)在生物体内具有抗氧化、抗衰老和重金属解毒等重要的生理功能。谷氨酸半胱氨酸连接酶(glutamate cysteine ligase,GCS)是合成谷胱甘肽的关键酶,又名γ-谷氨酰基半胱氨酸合成酶(γ-glutamylcysteine synthetase,γ-ECS)。本研究利用RT-PCR技术从甘蓝型油菜(Brassica napus L.)镉低积累品种‘美国油王88’中克隆到谷胱甘肽合成酶基因GCS的2个拷贝,分别命名为Bn-GCS-1和Bn-GCS-2。序列分析表明Bn-GCS-1的ORF长度为1536 bp,推测编码蛋白含有511个氨基酸,分子量为57.58 kD,等电点为6.02;Bn-GCS-2的ORF为1545 bp,推测编码514个氨基酸,分子量为57.95 kD,等电点为5.90。氨基酸序列比对和进化树分析发现,Bn-GCS-1和Bn-GCS-2与其他植物同源蛋白具有较高的一致性,且与白菜型油菜和甘蓝型油菜归为一类,亲缘关系最近。2个蛋白都由保守结构谷氨酸-半胱氨酸连接酶,γ-谷氨酰半胱氨酸合成酶组成,属于谷氨酰半胱氨酸连接酶家族基因。亚细胞定位预测结果表明定位在叶绿体的概率为99.8%。本研究结果为进一步分析Bn-GCS-1和Bn-GCS-2基因功能及谷胱甘肽生物合成提供了理论依据。Glutathione(GSH)has many important physiological functions such as antioxidation,antisenility and heavy metal detoxification.Glutamate cysteine ligase(GCS)is a key enzyme of glutathione synthesis in plants,also known asγ-glutamylcysteine synthetase(γ-ECS).In this study,two copies of the Bn-GCS genes were cloned from Brassica napus L.(American cultivar’Youwang88’of low Cd accumulation)by RT-PCR techniques,and designated as Bn-GCS-1 and Bn-GCS-2 respectively.Sequence analysis showed that the cDNA of Bn-GCS-1 contained a 1536 bp ORF encoding a protein of 511 amino acids with a predicted molecular weight of 57.58 kD and theoretical isoelectric point of 6.02;the c DNA of Bn-GCS-2 contained a 1545 bp ORF encoding a protein of 514 amino acids with a predicted molecular weight of 57.95 kD and theoretical isoelectric point of 5.90.Multiple sequences alignment and phylogenetic analysis indicated that the Bn-GCS-1 and Bn-GCS-2 shared high similarity with GSH homologous protein from other plants,belonged to the same branch with Brassica rapa subsp.Chinensisγ-ECS and Brassica napusγ-ECS and had the closest phylogenetic relationship with them.Both proteins with glutamic acid cysteine ligase and gamma glutamylcysteine synthetase conserved domains,indicate that Bn-GCS-1 and Bn-GCS-2 belong to glutamate cysteineligase modulatory family.Subcellular localization analysis showed that BnGCS-1 and Bn-GCS-2 proteins were located in chloroplast with 99.8%probability.The results provide a theoretical basis for further analysis of Bn-GCS-1 and Bn-GCS-2 genes function and glutathione biosynthesis.

关 键 词：油菜 GCS基因 克隆 生物信息分析 

分 类 号：S565.4[农业科学—作物学]