Cloning Of Antifreeze Protein Gene From Carrot And Genetic Transformation Of Grape

In China the freezing resistance of main grape cultivars is weak.Especially thedrought resistance capacity of root is poor so that it needs to be covered soil to over thewinter.Therefore,though genetic transformation, plant antifreeze protein will be introducedinto grapes.Developing the transgenic grape variety is important to improve the freezeresistance of the grapes.In the syudy,It succeeded to clone(AFP)by Polymerase ChainReaction(PCR),and having sequence analysis. A plant expression vector containingantifreeze protein(AFP)was constructed.Agrobacterium tumefaciens-mediated transfor-mation was used for genetic transformation of Ruby Seedless.The main results are asfollows:1. Genomic DNA was extracted form the seeding of carrot,which is the template, thecarrot antifreeze protein was cloned by Polymerase Chain Reaction (PCR),and it hasconnected into the pGEM-Teasy vector and named pGEM-AFP.2.According to the analysis of software of ProtParam and Predictprotein to bioinfor-mation,the AFP protein′s molecular formula was predicted,which is C1709H2680N448O502S16. The protein molecular weight was38.048kD and theoretical was5.43. The proteinencoding by AFP was hydrophilicity and secretory protein,and there was signal peptide.The secondary structure ofAFP protein mainly included α-helix, β-sheet and random coil.3.After pGEM-AFP and pBI121has been double digestion,they connectedtogether,then transform to competent cells from Escherichia coli (DH5α), and successed inconstructing the plant express vector(pBI-AFP).4. The plant express vector pBI-AFP was introduced into Agrobacterium tumefaciensEHA105by freezing-thawing method.By screening byantibiotic resistance and PCR toidentify the recombinant plasmid,the results confirmed assuredly,which called pBI-AFP/At.EHA105.5.Optimum bud regeneration from leaves of Ruby Seedless was obtained in CHmedium containing2-iP4mg/L,6-BA2mg/L and TDZ4mg/L,the regenerationg rates is53.3%；the medium for adventitious buds from leaves of Super Seedless was CH mediumcontaining2-iP4mg/L,6-BA2mg/L,TDZ0.5mg/L,CPPU0.5mg/L,ZT1mg/L,ration of adventitious buds is46.7%.6. With the agrobacterium-mediated transformation of grape leaf disc method, it has beenobtained transgenic plant of grape. Identification by PCR technology, it has been provedthat the carrot AFP gene is inserted in the grape plantets.