一个新的二色补血草金属硫蛋白基因LbMT2的克隆及其表达分析  

Cloning and expression of a novel metallothionein gene LbMT2 from Limonium bicolor

作　　者：班巧英[1] 刘桂丰[1] 王玉成[1] 张大伟[1] 蒋丽丽[1] 

机构地区：[1]东北林业大学,哈尔滨150040

出　　处：《遗传》2008年第8期1075-1082,共8页Hereditas（Beijing)

基　　金：教育部科学技术研究重点项目(编号:107037);国家自然科学基金面上项目(编号:30571509);黑龙江省攻关重点项目(编号:GB06B303-1)资助~~

摘　　要：从二色补血草cDNA文库中分离出一个新的金属硫蛋白基因LbMT2全长cDNA序列。该基因全长518bp,其中5′非翻译区(UTR)64bp,3′非翻译区205bp,开放阅读框(ORF)249bp,编码由82个氨基酸组成的蛋白质,编码蛋白的分子量为8.1kDa,理论等电点(pI)为4.72。利用实时定量PCR方法研究了二色补血草在CuSO4、CdCl2、NaCl、低温和PEG胁迫下不同时间该基因的表达模式。结果表明,CuSO4、CdCl2、NaCl和低温处理均能诱导LbMT2基因在二色补血草的根和叶中的表达,而PEG处理则抑制了LbMT2在根和叶中的表达。构建LbMT2基因的原核表达载体pGEX-LbMT2,通过IPTG诱导在大肠杆菌(Escherichia coli)BL21中融合表达,SDS-PAGE电泳获得35kDa的蛋白条带,大小与预期相符。The full length cDNA of a novel metallothionein （LbMT2） gene was cloned from a cDNA library of Limonium bicolor. The LbMT2 gene cloned is 518 bp in length, which includes a 64 bp of 5＇ untranslated region （UTR） and a 205 bp of 3＇ untranslated region. This gene has an open reading frame （ORF） of 249 bp in length, encoding a protein of 82 amino acid residues with the molecular mass of 8.1 kDa and theoretical pI of 4.71. The expression of LbMT2 gene in L. bicolor in response to CuSO4, CdCl2, NaCl, cold, and PEG was further investigated using real time quantitative PCR. In both leaf and root of L. bicolor, the expression of LbMT2 was induced by CuSO4, CdCl2, NaCl, and cold, but inhibited by PEG stress. LbMT2 gene was inserted into a prokaryotic expression vector （pGEX-4T-2） to produce the recombinant expression vector pGEX-LbMT2. The expression of LbMT2 in E. coli BL21 was induced with IPTG, which produced a protein band with expected size of 35 kDa on SDS-PAGE.

关 键 词：二色利 血草 金属硫蛋白 实时定量PCR 原核表达 

分 类 号：S54[农业科学—作物学]