PHD类转录因子GmPHD2和Bar基因的植物表达载体的构建

Construction of a Plant Expression Vector Containing both the PHD-type Transcription Factor Gene GmPHD2 and Bar Gene

利用高保真PCR酶从克隆载体pMD18T-GmPHD2中扩增出大豆PHD类转录因子GmPHD2,并于基因上下游分别引入XbaI和XhoI酶切位点;然后通过TA克隆、载体双酶切、连接、转化等技术手段,将其连入植物表达载体pBA002,构建了含GmPHD2和抗除草剂筛选标记Bar基因的植物表达载体pBA002-GmPHD2。重组质粒经PCR检测、双酶切及测序验证,表明GmPHD2基因已被完整、正确的插入到pBA002载体中,之后通过冻融法将重组质粒pBA002-GmPHD2成功导入农杆菌EHA105,利用此农杆菌不仅可以介导转化大豆,而且可以转化非同源的其他作物,为进一步开展植物抗逆性的基因工程研究提供了新基因资源。

The PHD-type transcription factor gene GmPHD2 was amplified from cloning vector pMD18T-GmPHD2 using high-fidelity PCR technology.The XbaI and XhoI restriction sites were introduced to the up-and downstream of the full length CDS,respectively.The plant expression vector pBA002-GmPHD2 which contained both the GmPHD2 gene and the selective marker Bar gene for herbicide resistance was accurately constructed through a series of molecular means,including TA cloning,double enzyme digestion,ligation,transformation and so on.The obtained vector was further verified by PCR amplification,enzyme digestion,and sequencing.Then,the vector pBA002-GmPHD2 was successfully transferred into Agrobacterium tumefaciens.The T-DNA of the Agrobacterium tumefaciens can be transformed into not only soybeans,but also other non-homologous crops,which offers a new genetic resource for improving the stress-tolerant ability of plants.