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植物溶血磷脂酸酰基转移酶(lysophospholipid acid acyltansferase,LPAT)是在植物不同组织中调控溶血磷脂酸生成各种磷脂酸的代谢流的关键酶。本研究采用同源克隆的方法,从甘蓝型油菜湘油15中克隆BnLPAT4基因翻译起始位点上游的调控序列,长度为1 326 bp。Plantcare在线分析表明:该序列含有CAAT-box和TATA-box等核心启动子原件,同时还有多个光响应元件、逆境胁迫响应元件、激素应答元件等。将该启动子与GUS基因融合,构建pBnLPAT4:GUS植物表达载体,通过农杆菌介导法获得拟南芥T3代转基因株系。GUS组织化学染色显示,幼苗期的转基因拟南芥在叶和根部均具有GUS活性,成熟期在莲座叶、花瓣、花萼、花托及果荚中表达,而花药及种子中未检测到GUS活性。
Lysophospholipid acid acyltansferase (LPAT) is a key enzyme that regulates the metabolism of lysophosphatidic acid to various phosphatidic acids in different tissues of plants. In this study, homologous cloning method was used to clone the upstream regulatory sequence of BnLPAT4 gene from the Brassica napus Xiang You 15 with a length of 1 326 bp. Plantcare online analysis shows that the sequence contains core promoter elements such as CAAT-box and TATA-box, as well as multiple light response elements, stress response elements and hormone response elements. The promoter was fused with GUS gene to construct pBnLPAT4: GUS plant expression vector, and the Arabidopsis thaliana T3 generation transgenic line was obtained by Agrobacterium-mediated transformation. GUS histochemical staining showed that transgenic Arabidopsis plants had GUS activity in both leaf and root at seedling stage, and expressed in rosette leaves, petals, calyx, receptacle and pod at maturity, while no GUS activity was detected in anthers and seeds.