Study On Cloning Transcription Factor CsCBF3and Genetic Transformation In Cucumber

Cucumber is an important vegetable in the world, as well as one of the major crops cultivated in greenhouse. Cucumber is one of typical chilling-sensitive plants, low temperature in the early spring and late autumn or winter is the most directly limited factor of cucumber yield and quality. Therefore, improving cold tolerance becomes the focus of year round cucumber supply, and breeding varieties highly resistant to low temperature is the major strategy. Genetic engineering can not only break the limitation of narrow genetic base and lacking of excellent genes of cucumber, but also has advantages such as shorter cycle of breeding, lower expense and stronger purpose. So transgenic technology provides a better and faster way to improve the cold tolerance of cucumber.Transcription factor CBF is a key’molecular switch’for plant to receive low temperature signal and regulate adaptive response. CBF could activate several downstream target functional genes relate to cold tolerance. Given to the important role CBF played in the process, it will be very helpful to determine its function in cucumber through studying the plant tolerance to low temperature, cloning cucumber CBF and investigating its expression characteristics.Transfering CBF into cucumber by transgenetic technology could improve cucumber tolerance to low temperature significantly, and also provide a new germplasm for cucumber cold tolerance breeding.Based on the background all above, cloning of cucumber CBF3and genetic transformation were carried out, concrete contents and results are as follows:1. Cloning and expression analysis of CsCBF3gene from Cucumis sativus L.In order to determine its role in cold response in cucumber(Cucumis sativus L.), CsCBF3was cloned from Chipper using RT-PCR method and its GenBank accession number was JQ900769. CsCBF3had an open reading frame of615bp, which was supposed to encode a protein of204amino acids. Phylogenetic tree analyses showed that CsCBF3belonged to CBF gene family, and had close relationship with CBF3from Vitis amurensis, while had far relationship with CBF3from Lolium perenne and Oryza sativa. Bioinformatics analysis showed that CsCBF3protein was consisted of AP2DNA binding domain which included amino acid residues, nuclear localization signal region and acidic activation domain, and some phosphorylation sites and protein secondary structures may play important roles in process of interactions between proteins and DNA. Quantitative real-time PCR results showed that expression of CsCBF3could be induced by low temperature, and the level reached to peak very fast and then decreased. These results suggested that CsCBF3was a rapid response gene and played an important role in resistance to low temperature in cucumber.2.Influence of acetosyringone and pH of co-culture medium on cucumber genetic transformation efficiencyCucumber genetic transformation efficiency affected by acetosyringone concentrations and co-culture pH were studied with cotyledonarynode of cucumber’Changchun mici’. The results indicated that both acetosyringone added in bacteria liquid and co-culture medium could significantly improve resistance bud induction rate, and the optimum AS concentration was100μmol/L. Cucumber resistance bud induction rate was also affected by different pH of co-culture medium and the most suitable pH for cucumber genetic transformation was5.2. These research results were helpful to optimize system of cucumber genetic transformation and increase transformation efficiency, as well as prompted transgenic work of cucumber.3.Construction of Expression Vector of CsCBF3and Transformation to CucumberCsCBF3was cloned using specific primers designed according to its ORF sequence and was inserted into pCAMBIA1304in forward orientation by double enzyme digestion. The recombinant plasmid was then introduced into Agrobacterium tumefaciens strain EHA105by freeze-thaw method. Electrophoresis results showed that recombinant expression vector pCAMBIA1304-CsCBF3was successfully constructed and introduced into EHA105. Cotyledon node of’Changchun mici’was transformed by Agrobacterium Mediated method, and hygromycin resistant plants were obtained after selection and domestication.CsCBF3was proved to be introduced into cucumber genome successfully by PCR detection with a transformation rate of4.8%.