银杏GbPAL基因启动子表达载体的构建

摘要

为弄清GbPAL启动子调控类黄酮代谢的功能,采用双酶切方法,用BamH Ⅰ+HindⅢ将GbPALp与植物表达载体pBI121分别酶切纯化,通过T4 DNA连接酶将GbPALp片段连接到已切除35S启动子的植物表达载体pBI121上,并转化到农杆菌LBA4404上,然后进行菌落PCR及酶切鉴定.结果表明:扩增到的GbPALp片段成功引入了BamH Ⅰ和HindⅢ酶切位点,GbPALp酶切后与酶切前的条带大小一致；将酶切后的空质粒和引入酶切位点的目的片段进行连接转化后,含目的基因pBI121:GbPALp:LBA4404菌落培养成功.成功构建了GbPAL基因启动子真核表达载体pBI121:GbPALp.%The GbPALp and plant expression vector pBI121 were digested with BamH I + Hind Ⅲ , respectively. Then the purified GbPALp fragment was connected to the plant expression vector pBI121 without 35S promoter by T4 DNA ligase. Finally the pBI121 containing GbPAL gene promoter was successfully transferred into Agrobacterium tumefaciens LBA4404. The transferred A. Tumefaciens LBA4404 with pBH2-.GbPALp was identified by PCR and enzyme digestion.