抗冻蛋白基因AFP表达载体构建及转化紫花苜蓿初报

以胡萝卜基因组DNA为模板,用PCR方法扩增目的基因,连接到PGEM-T Easy Vector载体上,经XbalⅠ和SacⅠ双酶切,将目的片段连接到PBI121工程质粒上,通过农杆菌介导法转化和田苜蓿,为提高紫花苜蓿的抗冻性奠定基础.结果表明,成功克隆出抗冻蛋白基因AFP,并构建成AFP植物表达载体.获得了具有卡那霉素抗性的苜蓿愈伤组织,和田苜蓿愈伤组织的最佳Kan筛选浓度为60 mg/L.经PCR技术鉴定,AFP抗冻蛋白基因已整合到苜蓿基因组中.

Construction of Antifreeze Protein Gene AFP Expression Vector and Transformation into Alfalfa Callus

In order to enhance the freezing tolerance of alfalfa, the carrot genomic DNA was taken as template, the target gene was amplified using PCR method and connected to the PGEM-T Easy Vector vector, the target fragment was connected to the engineering plasmid PBI121 after double digestion by Xbal Ⅰ and Sac Ⅰ, then alfalfa Hetian was transformed through Agrobacterium-mediated transformation. The results show that the antifreeze protein gene AFP was successfully cloned, AFP plant expression vector was constructed, and the kanamycin-resistant callus of alfalfa was obtained. The optimal kanamycin selection concentration of alfalfa Hetian callus was 60 mg·L~(-1). Based on the identification by PCR technology, AFP antifreeze protein gene was integrated into the alfalfa genome.