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作物学报 ›› 2012, Vol. 38 ›› Issue (10): 1856-1863.doi: 0.3724/SP.J.1006.2012.01856

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

花生γ-生育酚甲基转移酶基因(γ-TMT)的克隆及序列分析

李拴柱,万勇善*,刘风珍*   

  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
  • 收稿日期:2012-02-15 修回日期:2012-06-10 出版日期:2012-10-12 网络出版日期:2012-07-27
  • 通讯作者: 万勇善, E-mail: yswan@sdau.edu.cn, Tel: 0538-8241540; 刘风珍, E-mail: liufz@sdau.edu.cn, Tel: 0538-8241540
  • 基金资助:

    本研究由山东省花生良种产业化工程项目, 国家现代农业产业技术体系建设专项资金项目(CARS-14)和国家“十二五”科技支撑计划项目(2011BAD35B04)资助。

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

LI Shuan-Zhu,WAN Yong-Shan*,LIU Feng-Zhen*   

  1. State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, Shandong Agricultural University, Taian 271018, China
  • Received:2012-02-15 Revised:2012-06-10 Published:2012-10-12 Published online:2012-07-27
  • Contact: 万勇善, E-mail: yswan@sdau.edu.cn, Tel: 0538-8241540; 刘风珍, E-mail: liufz@sdau.edu.cn, Tel: 0538-8241540

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. hypogaea L.)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1 059 bp,编码352个氨基酸残基的AhTMT蛋白。AhTMT分子量为39.09 kD,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%、95.63%。

关键词: 花生, &gamma, -生育酚甲基转移酶, 基因克隆, 序列分析

Abstract:

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 combined with PCR from six cultivars and four diploid wild species that belong to Arachis section. cDNA sequence of γ-TMT (designated as AhrTMT) was cloned from Fenghua 2. Nucleotide sequencesof 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% random 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 homology of AhgTMT from cultivars and those from A. ipaensis (BB), A. batizocoi (BB), A. dura nensis (AA) and A. kuhlmannii (AA) was 100%, 99.91%, 99.74%, and 95.63%, respectively.

Key words: Peanut (Arachis hypogaea L.), Vitamin E, &gamma, -tocopherol methyltransferase, Gene cloning, Sequence analysis

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