中国农业科学 ›› 2012, Vol. 45 ›› Issue (14): 2904-2912.doi: 10.3864/j.issn.0578-1752.2012.14.013

• 园艺 • 上一篇    下一篇

平邑甜茶金属硫蛋白基因MhMT2的克隆和表达分析

 王顺才, 梁东, 马锋旺   

  1. 1.天水师范学院生命科学与化学学院,甘肃天水748100
    2.西北农林科技大学园艺学院,陕西杨凌712100
  • 收稿日期:2012-03-21 出版日期:2012-07-15 发布日期:2012-05-08
  • 通讯作者: 通信作者马锋旺,E-mail:fwm64@sina.com
  • 作者简介:王顺才,E-mail:wscai001@sina.com
  • 基金资助:

    国家“863”计划项目(2008AA10Z157)、甘肃省科技厅农业科技成果转化资金计划项目(1105NCNE109)

Cloning and Expression Analysis of Metallothionein Gene (MhMT2) in Malus hupehensis

 WANG  Shun-Cai, LIANG  Dong, MA  Feng-Wang   

  1. 1.天水师范学院生命科学与化学学院,甘肃天水748100
    2.西北农林科技大学园艺学院,陕西杨凌712100
  • Received:2012-03-21 Online:2012-07-15 Published:2012-05-08

摘要: 【目的】克隆平邑甜茶金属硫蛋白(metallothionein,MT)基因,探讨该基因对逆境的响应机理,为苹果抗逆分子育种提供依据。【方法】采用电子克隆和RT-PCR技术从平邑甜茶叶片中克隆MhMT2基因,利用生物信息学方法对获得的cDNA序列及推测氨基酸序列进行分析,通过定量PCR技术研究在胁迫条件和激素处理下该基因在叶片中的表达模式。【结果】平邑甜茶MhMT2基因cDNA全长534 bp,具有1个243 bp的完整开放阅读框,编码含80个氨基酸的多肽,其分子量为7.87 kD。同源性比对显示,MhMT2编码的氨基酸序列与其它植物的MT2蛋白有很高的相似性,其中与小金海棠的同源性最高,达到96.4%。MhMT2编码的多肽包含14个Cys残基,其N端富含Cys的结构域具有C-C、C-X-C和C-X-X-C基序,而C端仅有C-X-C基序。聚类分析显示,该基因属于植物MT2基因家族。定量PCR分析表明,MhMT2基因在叶中表达量最高,其次是根,在茎中表达量最低。在ABA、H2O2、机械损伤及NaCl处理下,该基因在叶片中的表达都上调,其中以H2O2最明显。【结论】对MhMT2基因在ABA、H2O2、机械损伤及盐胁迫下的表达模式分析,预示该基因表达水平在植物抗逆反应中起着重要作用。

关键词: 平邑甜茶, 金属硫蛋白基因, 克隆, 表达

Abstract: 【Objective】The objective of this study is to clone the full-length cDNA of metallothionein (MT) gene from rootstock (Malus hupehensis), to understand the mechanism of MT in response to environmental stress and provide fundamental evidence for molecular breeding of apple trees under stress conditions.【Method】The full-length cDNA sequence of MhMT2 gene was amplified from the leaves of M. hupehensis using in silico cloning and RT-PCR. Bioinformatics methods were used to analyze cDNA sequence obtained and putative amino acid sequence. The expression profiles of MhMT2 transcript in control seedlings and those exposed to various abiotic stresses and hormone treatments were detected by quantitative real-time RT-PCR.【Result】Sequence analysis indicated that MhMT2 cDNA was 534 bp in length and contained an intact open reading frame of 243 bp, encoding a polypeptide of 80 amino acids with a theoretical molecular mass of 7.87 kD. Homology comparison analysis showed that the deduced MhMT2 protein was highly homologous to other MT2 proteins from plant species, sharing a 96.4% homology with M. xiaojinensis. Bioinformatic analysis revealed that the putative MhMT2 protein contained 14 cysteine residues occurring as C-C, C-X-C, and C-X-X-C motifs in the N-terminal, and of C-X-C in the C-terminal region (X are other amino acids other than cysteine). Phylogenetic analysis suggested that this gene belonged to type 2 plant MT genes family. Quantitative real-time RT-PCR analysis indicated that MhMT2 showed the highest transcript abundance in leaves, moderate level in roots and the lowest level in shoots. MhMT2 transcripts in leaves were markedly increased by abscisic acid (ABA), hydrogen peroxide (H2O2), mechanical wounding, and NaCl, especially H2O2 treatment.【Conclusion】The induced expression profiling of MhMT2 gene under ABA, H2O2, mechanical wounding, and salt stress indicates that the MhMT2 gene may play an important role in response to these abiotic stresses.

Key words: M. hupehensis, metallothionein gene, cloning, expression