一个新的二色补血草金属硫蛋白基因LbMT2的克隆及其表达.pdfVIP

HEREDITAS (Beijing) 2008 8 , 30(8): 1075―1082 ISSN 0253-9772 研究报告 DOI: 10.3724/SP.J.1005.2008.01075 LbMT2 班巧英,刘桂丰,王玉成,张大伟,蒋丽丽 , 150040 : 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518 bp, 其中5′非翻译区(UTR)64 bp, 3′非翻译区205 bp, 开放阅读框(ORF)249 bp, 编码由82 个氨基酸组成的蛋白质, 编码蛋白的分子量为 8.1 kDa, 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 、 CdCl 、NaCl 、低温和PEG 胁迫下不同时间该基因的表达模式。结果表明, CuSO 、CdCl 、NaCl 和低温处理均 2 4 2 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电泳获得35 kDa 的蛋白条带, 大小与预期相符。 : 二色补血草; 金属硫蛋白; 实时定量PCR; 原核表达 Cloning and expression of a novel metallothionein gene LbMT2 from Limonium bicolor BAN Qiao-Ying, LIU Gui-Feng, WANG Yu-Cheng, ZHANG Da-Wei, JIANG Li-Li Northeast Forestry University , Harbin 150040, China Abstract: 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 CuSO , CdCl , NaCl, cold, and PEG was further investigated using real time quantitative PCR. In both leaf and 4 2 root of L. bicolor, the expression of LbMT2 was induced by CuSO , CdCl , NaCl, and cold, but inhibited by PEG stress. 4 2 LbMT2 gene was inserted into a prokaryotic expression vector (pGEX-4T-2) to p