小麦γ-TMT基因的克隆与表达分析  

Cloning and Expression Analysis of γ-Tocopherol Methyltransferase (γ -TMT) Gene in Wheat (Triticum aestivum)

作　　者：刘媛媛 郭启平 党仁美 张珊 闻珊珊 LIU Yuanyuan;GUO Qiping;DANG Renmei;ZHANG Shan;WEN Shanshan(College of Agronomy, Northwest A&F University/Laboratory of Molecular Biology and Biotechnology in Wheat, Yangling, Shaanxi 712100, China)

机构地区：[1]西北农林科技大学农学院/小麦分子生物学与生物技术实验室

出　　处：《西北植物学报》2019年第5期886-895,共10页Acta Botanica Boreali-Occidentalia Sinica

基　　金：西北农林科技大学学科建设项目“作物转基因平台建设”

摘　　要：γ-生育酚甲基转移酶(γ-TMT)是维生素E合成途径的关键酶之一。该研究采用RT-PCR方法,从小麦品种‘中国春’中克隆得到小麦γ-TMT基因的3个同源拷贝,其CDs(codingsequence)序列相似性为99.12%;其中Ta-γ-TMT-6A和Ta-γ-TMT-6B编码区长度为1101bp,编码366个氨基酸,而Ta-γ-TMT-6D因发生无义突变,编码365个氨基酸。生物信息学分析表明,3个蛋白分子量分别为39.58、39.53和39.50kDa,理论等电点分别为6.67、6.41和7.08,都属于亲水性不稳定非分泌型蛋白。系统进化分析表明,小麦Ta-γ-TMT蛋白与糜子Pm-γ-TMT的亲缘关系相对较近。实时定量PCR分析表明,小麦Ta-γ-TMT基因在根、茎、叶、颖壳、种子中均有表达,在叶片中表达量最高;ABA、NaCl、PEG均能诱导Ta-γ-TMT基因的上调表达。该研究结果为进一步探讨小麦γ-TMT基因的功能奠定了基础。Gamma-tocopherol methyltransferase (γ-TMT) is one of the key enzymes in the vitamin E synthesis pathway. In this study, we cloned three homologous copies of the γ-tocopherol methyltransferase gene (γ-TMT ) from the wheat cultivar-Chinese Spring using RT-PCR (reverse transcription PCR). The similarity of three sequence CDs (coding sequence) was 99.12%. Ta-γ-TMT-6A and Ta-γ-TMT-6B coding sequences both contain 1 101 bp and encode 366 amino acids, while Ta-γ-TMT-6D encodes only 365 amino acids due to nonsense mutations. The results of bioinformatics analysis showed that the molecular weights of the three proteins were 39.58, 39.53 and 39.50 kDa, respectively, and the theoretical isoelectric points were 6.67, 6.41 and 7.08. They all belonged to hydrophilic unstable non-secretory proteins. Phylogenetic tree analysis indicated that Ta-γ-TMT protein shared high similarity to γ-TMT protein from Panicum miliaceum and closest genetic relationship. Real-time quantitative PCR analysis showed that the Ta-γ-TMT gene was expressed in roots, stems, leaves, glumes and seeds, and highly expressed in leaves. Meanwhile, expression of the Ta-γ-TMT gene was up-regulated induced by ABA, NaCl, PEG treatments. This result will provide the foundation for further study of the function of the Ta-γ-TMT gene in wheat.

关 键 词：小麦 Γ-生育酚甲基转移酶 基因克隆 表达分析 

分 类 号：S512.1[农业科学—作物学] Q943.2[生物学—植物学]