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

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.