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.