龙眼胚性愈伤组织γ-ECS基因的克隆及表达分析

摘要

y-ECS基因编码的γ-谷氨酰半胱氨酸合成酶是细胞内谷胱甘肽(GSH)从头合成的限速酶.以龙眼转录组数据库为基础,采用同源克隆的方法从龙眼胚性愈伤组织中分离得到y-ECS基因cDNA全长序列,在GenBank上的登录号为JF815477.y-ECS基因序列全长1 812 bp,包括17 bp 5'UTR及254 bp 3'UTR; ORF长度为1 541 bp编码511个氨基酸.生物信息学分析结果表明,龙眼胚性愈伤组织γ-ECS的核甘酸序列及其推导的氨基酸序列分别与蓖麻、毛果杨等具有较高的同源性；γ-ECS为1个具有跨膜结构的非分泌蛋白,具有亲水性;亚细胞定位于过氧化物酶体,不含信号肽,并存在亮氨酸拉链结构.龙眼胚性愈伤组织y-ECS基因在龙眼体胚发生过程中的整体表达趋势是先升高,后降低,其表达量在心形胚阶段达到最大值之后在鱼雷形胚阶段急剧下降.y-ECS基因表达量变化可能与GSH在龙眼体胚发生过程中的含量变化一致.%Glutathione (γ-glutamylcysteinyl-glycine)is synthesized de novo through two steps,and gammaglutamyleysteine synthetase(γ-ECS)is a rate-limiting enzyme in GSH biosynthesis.Based on the database of longan transcriptome,the γ-ECS gene(Gen Bank accession No.JF815477)from longan embryogenic callus was obtained by the homologous cloning and RACE technology.Full length cDNA sequence of γ-ECS was 1 812 bp,encoding 511 amino acids,including a 5' untranslated region of 17 bp and a 3'untranslated region of 254 bp and an open reading frame of 1 541 bp.Bioinformatics analyses showed that both the nucleotides and amino acids sequences of the γ-ECS gene were highly homologous with those of other species.The γ-ECS was a transmembrane protein.It was hydrophilic and without a signal peptide.The subcellular localization of γ-ECS protein was in the peroxisome and it had a leucine zipper structure.The expression level of γ-ECS gene during somatic embryogenesis reached the maximum in heart-shape embryo and then declined sharply in torpedo-shape embryo.The expression level changes of γ-ECS gene may be consistent with the content of GSH during longan somatic embryogenesis.