花生γ-生育酚甲基转移酶基因(γ-TMT)的克隆及序列分析  

Cloning and Bioinformatic Analysis of γ-Tocopherol Methyltransferase Gene (γ-TMT) in Peanut

作　　者：李拴柱[1] 万勇善[1] 刘风珍[1] 

机构地区：[1]山东农业大学/作物生物学国家重点实验室/山东省作物生物学重点实验室,山东泰安271018

出　　处：《作物学报》2012年第10期1856-1863,共8页Acta Agronomica Sinica

基　　金：山东省花生良种产业化工程项目;山东省现代农业产业技术体系花生创新团队建设项目;国家现代农业产业技术体系建设专项资金项目(CARS-14);国家"十二五"科技支撑计划项目(2011BAD35B04)资助

摘　　要：γ-生育酚甲基转移酶（γ-TMT）催化γ-生育酚转变为生物活性最高的α-生育酚,是决定植物中维生素E成分和活性的关键酶。本研究采用电子克隆与PCR相结合的方法获得了花生6个栽培品种（A.hypogaeaL.）和花生区组4个二倍体野生种中γ-TMT的全长DNA序列,定名为AhgTMT;从栽培品种丰花2号中获得γ-TMT的cDNA序列,定名为AhrTMT。同一栽培品种的AhgTMT与AhrTMT的序列完全对应,说明该基因无内含子。AhrTMT编码区长1059bp,编码352个氨基酸残基的AhTMT蛋白。AhTMT分子量为39.09kD,等电点6.72,总平均亲水性–0.12;预测的二级结构中α-螺旋占55.40%,β-折叠占10.51%,无规则卷曲占34.09%;被定位于叶绿体,含有甲基转移酶保守的SAM结构域。AhTMT与已报道植物γ-TMT氨基酸序列的相似性为61.75%~72.80%。栽培品种的AhgTMT与A.ipaensis（BB）、A.batizocoi（BB）、A.duranensis（AA）、A.kuhlmannii（AA）4个野生种的核苷酸序列同源性分别是100%、99.91%、99.74%和95.63%。γ-tocopherol methyltransferase（γ-TMT） is one of the critical enzymes that determine the composition and activity of vitamin E in plant.γ-TMT catalyzes the biological reaction from γ-tocopherol to α-tocopherol, is the most bioactive component of vitamin E. In this study, DNA sequence of γ-TMT in peanut （designated as AhgTMT ） was cloned by using in silico cloning com- bined with PCR from six cultivars and four diploid wild species that belong to Arachis section, cDNA sequence of γ-TMT （desig- nated as AhrTMT） was cloned from Fenghua 2. Nucleotide sequences of AhgTMT are identical with AhrTMT, which means no intron in this gene. AhrTMT has a 1 059 bp open reading frame encoding a protein （AhTMT） of 352 amino acid residues with a molecular weight of 39.094 kD. The isoelectric points of AhTMT is 6.72 and its grand average of hydropathy is -0.12. Secondary structure prediction of AhTMT indicated that 55.40% of the protein sequence is alpha helixes, 10.51% beta turn and 34.09% ran- dom coil. AhTMT has a conserved SAM domain and is localized in chloroplasts. The similarity between AhTMT and γ-TMTs reported in other species is from 61.75% to 72.80%. Alignment analysis of AhgTMT nucleotide sequences indicated that the ho- mology of AhgTMT from cultivars and those from A. ipaensis （BB）, A. batizocoi （BB）, A. duranensis （AA） and A. kuhlmannii （AA） was 100%, 99.91%, 99.74%, and 95.63%, respectively.

关 键 词：花生 维生素E Γ-生育酚甲基转移酶 基因克隆 序列分析 

分 类 号：S565.2[农业科学—作物学]