Genetic Transformation Of CRT/DRE Binding Factor CBF1 In Hairy Tomato (Lycopersicon Hirsutum)

Tomato is an important vegetable crops which is cultivated worldwide, but it frequently suffers from the cold damage so as to cause the economic benefits loss, affecting tomato production in the productive process. The Lycopersicon hirsutum is a wild tomato, which belongs to Lycopersicon genus. Previous studies have shown that it is tolerant to low temperatures and even freezing.Through the observation of hairy tomato leaves, its surface has the thick dense hair, but cultivated tomatoes do not have this characteristic. It is postulated that the surface of hairy tomato may improve the ability of its cold tolerance to a certain extent. In addition, the enhancement of cold tolerance of hairy tomato may be improved the regulation of cold tolerance, such as the degree of membrane lipid phase transition is small, the adjustment of membrane structure can quickly adapt to low temperatures, metabolic adjustment and a number of physiological and biochemical changes. It is postulated that hairy tomato itself may have its unique and powerful low temperature tolerant gene.At present, the research of plant cold-resistance gene was mainly concentrated in the anti-cold transcriptional factor CBFgenes. In cold conditions, the cells suffer severe dehydration. At first plant cell membrane is damaged. When the plant met cold stress, the expression of cold tolerance-related gene which is regulated by CBFgene prevented the cell membrane lipid bilayer to form hexagonal II phase lipid changes and cell dehydration caused by the low-temperature.The surface of hairy tomato has thick dense hair coverage, while Solanum lycopersicoides has narrow and thin leaves, so we speculated that regulation of CBF1 gene is the dominant ways of cold-tolerance mechanisms in Lycopersicon esculentum and Solanum lycopersicoides.1. In this study, cultivated tomato Jinpeng 1, cold-resistant cultivated tomato L402 and hairy tomato as materials. Under the condition of 5℃low temperature, compared with the effect of physiological and biochemical mechanisms between hairy tomato(Lycopersicon hirsutum) and cultivated tomato(Lycopersicon esculletum) in the diffierent treatment time. Three kinds of tomatoes are treated with 5℃low temperature and their physiological and biochemical characteristics were tested, and then cultivate tomato as the control.(1)With processing time, malondialdehyde (MDA) content and superoxide radical (O2-) of Lycopersicon hirsutum were significantly lower than that of cultivated tomatoes, while the activities of superoxide dismutase (SOD) activity, peroxidase (POD) activity, proline content, catalase (CAT) activity, ascorbate peroxidase (ASA) and phenylalanine ammonia-lyase (PAL) were significantly higher than that of cultivated tomatoes. (2)After 24h of low temperature treatment, no obvious change was found in leaves of hairy tomato, but leaves of cultivated tomato cultivars showed wilting symptom.2. Based on the above study, this study take the CBF1 gene as materials, it constructs into the vector and achieves as the following results.(1)plant expression vectors pCAMhp1033CBF1 and pRDhp1033CBF1 which had respectively strong constitutive CaMV35S and cold-inducible Rd29A promoter to regulate the Lycopersicon hirsutum CBF1 gene were constructed.(2)plant expression vectors pCAMhp2408CBF1 and pRDhp2408CBF1 which had respectively strong constitutive CaMV35S and cold-inducible Rd29A promoter to regulate the Solanum lycopersicoides CBF1 gene were constructed.3. By Agrobacterium-mediated method, CBF1 gene has been transfered into Micro-tom tomato.(1)we gained 19 regenerated plants, which 13 were moved into soil and 6 are not moved into the soil through continued regeneration development. We detected the 13 plants which moved into soil by PCR molecular detection and gained 8 positive plants.(2)The patience of transgenic plants was tested at low temperatures. With the extension of processing time, the result of physical and chemical analysis demonstrated that transgenic tomato plants' superoxide dismutase (SOD) activity, peroxidase (POD) activity, proline content, catalase (CAT) activity, ascorbate peroxidase (ASA) activity and phenylalanine ammonia-lyase (PAL) activity were significantly higher than cultivated tomatoes. while malondialdehyde (MDA) content and superoxide radical (O2-) were significantly lower than cultivated tomatoes.