抗冻基因CBF_2表达载体构建及转化紫花苜蓿的研究

Construction of antifreeze gene CBF_2 expression vector and transformation into alfalfa callus

本研究以拟南芥基因组DNA为模板,用PCR方法扩增目的基因,连接到PGEM-T Easy Vector载体上构建成克隆载体T-CBF2。用BamHⅠ和SacⅠ分别对克隆载体T-CBF2和植物表达载体PBI121进行双酶切,获得目的片段和线性质粒。在T4DNA连接酶的作用下进行定向连接,构建成植物表达载体P-T-CBF2。采用直接转化法将重组子导入根癌农杆菌EHA105。经PCR鉴定,重组质粒已成功导入根癌农杆菌中。通过农杆菌介导法转化和田苜蓿,现在已经得到转基因的苜蓿愈伤组织。

Arabidopsis thaliana genomic DNA was selected as a template to amplify the target gene by PCR and connect it to the PGEM-T Easy Vector for construction of the T-CBF2.The target fragment and linear plasmids were obtained from the cloning vector T-CBF2 and from the plant expression vector PBI121 with dual digestion using BamHⅠand Sac I,respectively.The plant expression vector P-T-CBF2 was built through directional connections using T4 DNA ligase.PCR identification proved that the recombinant had been transferred into Agrobacterium tumefaciens and it was then introduced into Medicago sativa cv.Hetian through Agrobacterium-mediated transformation.Transfer of the target gene to alfalfa callus was successful.