Construction Of Gene AFP,CBF2 Expression Vector And Transformation Into Alfalfa Callus

Chilling injury of plant is one of the most serious natural disasters to agriculture and forestry production in the world,which can cause heavy yield loss to the global crops about hundreds of billions of dollars every year.Alfalfa(Medicago sativa) is an important warm-temperate grass, sensitive to temperature. Restricting its growth temperature is a major limiting factor, how to improve the cold resistance of alfalfa to improve their productivity has an important significance. The use of modern biological technology, molecular improvement of plant freezing tolerance is effective and targeted .In this study,the antifreeze genes AFP,CBF2 was cloned from carrot and Arabidopsis successfully, the plant expression vector was constructed and transferred into agrobacterium tumefacien.In view of the current problem of low conversion rate via Agrobacterium-mediated transformation method, and the optimum of alfalfa genetic transformation system was selected.The plant expression vector of antifreeze genes AFP,CBF2 was attempted to transform into an excellent alfalfa variety by using Agrobacterium-mediated.The results were as follows:1. Respectively carrot and Arabidopsis genomic DNA as a template, By PCR method, the full-length 1200bp of the antifreeze protein gene AFP and the full-length 700bp of antifreeze gene CBF2 were cloned successfully, Transformed into E.coli DH5α,after IPTG induction,the expression of antifreeze gene in the recombinant strain was detected.2. By double digestion, to the respective projects to connect to the PBI121 plasmid,to construct plant express vector P-T-AFP and P-T-CBF2 which was selected noting by Kanamycin.Then the new vector was further transferred into EHA105 by Direct transformation.3. Establishment of transformation system.Cultivated 7 days Cotyledon of M.sativa cv.HeTian as explants and agrobacterium tumefaciens strain as mediator,preculture 3 days,the infection time is 10 min, the agrobacterial density is OD=0.3,coculture 3 days,the Kanamycin selective pressure is 60 mg/L,400 mg/L Carb could restrained growing of agrobacterium.4. Through the transformation of alfalfa leaf disc method, respectively, AFP and CBF2 antifreeze genes into the genome of alfalfa, Identification by Kan and PCR technology, antifreeze gene AFP and CBF2 were integrated into the alfalfa genome.