铝胁迫下柱花草SSH文库构建及表达序列标签分析  

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

作　　者：王奇峰[1] 易琼[1] 李昆志[1] 陈丽梅[1] 玉永雄[2] 

机构地区：[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

出　　处：《植物学报》2010年第6期679-688,共10页Chinese Bulletin of Botany

基　　金：973计划(No.2007CB108901)

摘　　要：柱花草栽培种热研2号(Stylosanthes guianensis‘Reyan2’)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300μmol·L-1铝胁迫下正向cDNA文库。挑选插入片段大于300bp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。SSHcDNA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。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 AI-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.

关 键 词：铝胁迫 cDNA文库 表达序列标签 柱花草 抑制消减杂交 

分 类 号：S54[农业科学—作物学] Q943.2[生物学—植物学]