二穗短柄草BdGPX4基因的克隆及酶学性质
Cloning and characterization of glutathione peroxidase 4 from Brachypodium distachyon

为解析模式植物二穗短柄草中谷胱甘肽过氧化物酶（Glutathione peroxidase，GPX）在抗氧化过程中的作用机制，对二穗短柄草BdGPX4基因进行克隆，获得681 bp的cDNA序列，该基因编码226个氨基酸残基的蛋白质，预测相对分子量是24.49 ku。通过亚细胞定位预测，BdGPX4蛋白位于叶绿体和线粒体中。生物信息学分析发现BdGPX4基因启动子具有植物激素、光信号、干旱以及冷胁迫响应元件。结合二穗短柄草基因芯片数据和RT-PCR表达检测结果，发现BdGPX4基因在根、茎、叶、叶鞘、苗芽、穗状花序和种子等部位均表达，在光合组织和生长旺盛部位的表达显著高于非光合和衰老部位，并在根部受到SA诱导表达上调。成功构建BdGPX4基因的原核表达载体，在大肠杆菌中表达并纯化BdGPX4蛋白，检测到对H2O2和COOH具有较高活性，但对底物tBOOH活性较低，与直系同源蛋白OsGPX4相比功能发生分化。基因表达模式及酶学活性的特点预示着二穗短柄草BdGPX4基因在光合器官和胁迫条件下具有重要的抗氧化作用。
In order to analyze the antioxidative mechanism of glutathione peroxidase (GPX) in Brachypodium distachyon, a 681 bp cDNA of BdGPX4 gene is cloned, which encodes a protein of 261 amino acids residues with predicted relative molecular mass of 24.49 ku. The protein is presumably localized to the chloroplasts and mitochondria. Bioinformatics analysis show that the promoter of BdGPX4 gene contains many cis-acting elements related with stress such as cold, drought as well as plant hormone and light. Combining microarray data and RT-PCR expression results, BdGPX4 gene is found expressing in root, stem, leaf, leaf sheath, sprouts, spikes and seeds. Its expression in photosynthetic tissues and the active growing organs are significantly higher than that of non-photosynthetic and senescence parts. BdGPX4 is also induced by SA in roots. Recombinant plasmid is successfully constructed and the recombinant protein is purified. BdGPX4 protein shows high enzymatic activities towards the substrates H2O2 and COOH, and low enzymatic activities towards tBOOH. BdGPX4 protein is functionally differentiated compared to the orthologous protein OsGPX4 in rice. The characterizations of its expression patterns and enzyme activities indicate that the BdGPX4 gene plays an important antioxidative role in photosynthetic organs and under stress conditions.