植物学报 ›› 2010, Vol. 45 ›› Issue (06): 679-688.DOI: 10.3969/j.issn.1674-3466.2010.06.005

• 研究报告 • 上一篇    下一篇

铝胁迫下柱花草SSH文库构建及表达序列标签分析

王奇峰1, 易琼1, 李昆志1, 陈丽梅1*, 玉永雄2   

  1. 1昆明理工大学生命科学与技术学院生物工程技术研究中心, 昆明 650224
    2西南大学动物科技学院, 重庆 400716
  • 收稿日期:2010-05-24 修回日期:2010-07-05 出版日期:2010-11-01 发布日期:2010-09-20
  • 通讯作者: 陈丽梅

Construction of a Suppression Subtractive Hybridization Library for Stylosanthes guianensis Under Aluminum Stress and Expressed Sequence Tag Analysis

Qifeng Wang1, Qiong Yi1, Kunzhi Li1, Limei Chen1*, Yongxiong Yu2   

  1. 1Bioscience and Biotechnology Research Center, Kunming University of Science and Technology, Kunming 650224, China

    2College of Zoological Science and Technology, Southwest University, Chongqing 400716, China
  • Received:2010-05-24 Revised:2010-07-05 Online:2010-11-01 Published:2010-09-20
  • Contact: Limei Chen

摘要: 柱花草栽培种热研2号(Stylosanthes guianensis ‘Reyan 2’)对铝毒有较强的耐受性。为了鉴定其在铝胁迫下的诱导 基因, 利用抑制消减杂交(SSH)技术构建在300 μmol·L–1铝胁迫下正向cDNA文库。挑选插入片段大于300 bp的600个克隆进行测序, 共获得504条表达序列标签(EST)。序列重复性分析表明, 其中12.1%的EST只有1次重复, 61.4%的EST有2–16次重复, 重复出现次数较高的EST是细胞色素P450(53次, 占10.5%)、病原诱导型胰蛋白酶抑制剂(44次, 占8.7%)和衰老相关蛋白(37次, 占7.3%)。BLASTX分析显示, 504条EST中有97种非冗余基因, 其中包括46条功能已知基因和51条功能未知序列。46条功能已知EST中有30个为已报道铝胁迫相关基因, 16个是新发现的铝胁迫相关基因。SSH cDNA文库提供的信息为阐明柱花草耐铝毒的分子机制提供了重要线索。

Abstract: Stylo (Stylosanthes guianensis) is a forage grass grown worldwide. A cultivar of stylo (‘Reyan No.2’) was shown to be able to exudate citrate and tolerate aluminum (Al). To identify Al-induced genes in stylo, we constructed a forward cDNA library for Reyan No.2 under 300 μmol·L–1 Al stress by suppression subtractive hybridization. Approximately 600 clones with inserts > 300 bp were selected for sequence analysis. In total, 504 expressed sequence tags (ESTs) were obtained: 12.1% were singletons and 61.4% presented as 2–16 copies. The highly repetitive ESTs were 10.5% for cytochrome P450-like TBP protein, with 53 copies; 8.7% for pathogen-inducible trypsin-inhibitor-like protein, with 44 copies; and 7.3% for putative senescence-associated protein, with 37 copies. BLASTX analysis revealed that the 504 ESTs could be assembled into 97 non-redundant sequences, including 46 genes with known function. Of 46 genes with known function, 30 were previously reported to be Al-responsive genes and the other 16 ESTs are novel Al-associated genes. The information provides important clues to facilitate understanding the Al detoxification mechanism in stylo at the molecular level.