蓝猪耳胚珠磷脂酶C基因TfPLC1启动子的克隆及载体构建

应用衔接头PCR技术，以蓝猪耳全基因组DNA分别经DraI、EcoRV、PvuII、SmaI消化后与衔接头连接产物为模板，用衔接头引物和TfPLC1基因的特异引物经过多轮的巢式PCR，先后克隆到两个大小为798bp、813bp的TfPLC1基因上游序列；经测序、blastn比较分析和拼接得到一个蓝猪耳TfPLC1基因的启动子序列，共1432bp。序列分析表明它含有类似于TATA box和CAAT box的元件，在其远端上游区域还有多个AT富含区，而且还含有多个胚乳特异表达启动子的元件。将该启动子全序列和5’端缺失的700bp序列与PBI121分别构建了植物表达载体PPP1326和PPP700，用于转化蓝猪耳，以验证该启动子的功能。该启动子的克隆对于研究蓝猪耳胚珠TfPLC1基因的表达调控及功能具有重要意义。

Cloning and vector construction of ovule phospholipase C gene TfPLC1 promoter in Torenia fournieri

Torenia fournieri genomic DNA was digested with DraI、EcoRV、PvuII、SmaI respectively. A special adaptor was ligated to the ends of the digested DNA fragments as a template for adaptor PCR. With the adaptor and TfPLC1 gene-specific primers, bands of 798bp and 813bp upstream of TfPLC1 were obtained successively. After sequenced, blastn and combined, a TfPLC1 promoter of 1432bp was gained. TATA box and CAAT box-like elements were found in the sequence, and TA domain was rich in the far upstream of the promoter. And also some endosperm-specific elements were found in the sequence. PBI121 and the promoter were double digested to construct plant expression vectors PPP1326 and PPP700 by 5’ deletion. Then the vectors were transformed to Torenia fournieri to validate the function of the promoter.