中国农业科学 ›› 2013, Vol. 46 ›› Issue (9): 1857-1866.doi: 10.3864/j.issn.0578-1752.2013.09.013

• 园艺 • 上一篇    下一篇

苹果NADP依赖的苹果酸酶基因克隆、序列和表达分析

 董庆龙, 王海荣, 安淼, 余贤美, 王长君   

  1. 山东省果树研究所,山东泰安 271000
  • 收稿日期:2012-10-23 出版日期:2013-05-01 发布日期:2013-03-01
  • 通讯作者: 通信作者余贤美,Tel:0538-8299912;E-mail:yuxianmei95@163.com;通信作者王长君,Tel:0538-8266619;E-mail:wangcj@sdip.cn
  • 作者简介:董庆龙,Tel:0538-8299912;E-mail:dong19850412@163.com。通信作者余贤美,Tel:0538-8299912;E-mail:yuxianmei95@163.com;
  • 基金资助:

    山东省果树研究所所长科研基金(2012KY06)、国家自然科学基金项目(31100086)

Cloning, Sequence and Expression Analysis of NADP-Malic Enzyme Genes in Apple

 DONG  Qing-Long, WANG  Hai-Rong, AN  Miao, YU  Xian-Mei, WANG  Chang-Jun   

  1. Shandong Institute of Pomology, Tai’an 271000,Shandong
  • Received:2012-10-23 Online:2013-05-01 Published:2013-03-01

摘要: 【目的】克隆苹果(Malus×domestica B.)中苹果酸代谢的关键酶基因MdNADP-ME,进行序列特征分析,研究MdNADP-ME在苹果中组织表达的情况。【方法】利用RT-PCR技术获得苹果MdNADP-ME1,2,3全长的cDNA序列。对该序列进行生物信息学分析,采用荧光实时定量PCR技术研究MdNADP-ME1,2,3的组织表达。【结果】测序结果显示,获得的3个NADP-苹果酸酶基因(MdNADP-ME1、MdNADP-ME2 和 MdNADP-ME3; GenBank 登录号为JX971883、JX971884和JX971885),其ORF分别为1 512、1 782和1 926 bp,推测其分别编码503、593和641个氨基酸的多肽。氨基酸序列和结构分析显示,这3个基因均含有5个保守的氨基酸区域(motif I-V),含有2个功能结构域:malic和NAD_bind_1_malic_enz。进化树分析结果显示,MdNADP-ME1 和MdNADP-ME2属于双子叶植物细胞质NADP-ME型,MdNADP-ME3属于双子叶植物质体NADP-ME型。荧光实时定量PCR结果表明,MdNADP-ME1-3在被检测的组织中均有表达,但表达差异明显。【结论】MdNADP-ME1-3属于植物NADP-ME家族,结构高度保守,并且在苹果的不同组织中有不同表达模式。

关键词: 苹果 , 苹果酸酶 , 克隆 , 序列分析 , 表达分析

Abstract: 【Objective】This study is aimed to characterize the genes of MdNADP-MEs involved in malic acid metabolism in Malus×domestica B. by gene cloning, sequence and expression of MdNADP-MEs in different tissues.【Method】 Three full-length cDNA sequences of MdNADP-ME1,2,3 were isolated by RT-PCR. The obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods. qRT-PCR was used to assess the expression of MdNADP-MEs in different tissues.【Result】The sequencing results showed that three cDNAs (designated as MdNADP-ME1, 2 and 3; GenBank Accession No. JX971883, JX971884 and JX971885) were 1 512 bp, 1 782 bp and 1 926 bp. They contained an open reading frame (ORF) of 1 512 bp, 1 782 bp and 1 926 bp, respectively, and their ORFs encoded an protein with 503, 593 and 641 amino acids, respectively. Amino acid sequence and structure analysis indicated that MdNADP-MEs contained five conservative amino acid areas (motif I-V) and two functional structure domains: malic and NAD_bind_1_malic_enz. The result of phylogenetic analysis showed that MdNADP-ME1 and 2 belonged to cytosolic dicot NADP-ME (group I), MdNADP-ME3 belonged to plastidic dicot NADP-ME. qRT-PCR result showed that MdNADP-MEs were constitutively expressed in all examined tissues, but showed different expression levels.【Conclusion】MdNADP-ME1,2,3 belong to NADP-ME family, possess highly conserved structures, and show different expression patterns in different tissues of apple.

Key words: apple , malic enzyme , cloning , sequence analysis , expression analysis