Transformation Of Tobacco And Physiological Detection Of CBF1 Gene From Tomato

Solanum(Solanum lycopersicoides) is Solanum(Solanum) Tomato(Lycopersicon esculentum Mill) wild relatives, under the conditions of-1.25 ~ 5.3 ℃of it normal to fruition. Tobacco(Nicotiana tabacum L.) Solanaceae(Solanleae) Nicotiana(Nicotiana), one of the major Chinese tobacco as a cash crop, cryogenic freezing has restricted the development of production of high quality tobacco. CBF1 CBF gene family is a chilling transcription factor, it was first cloned in Arabidopsis. It can induced hypothermia regulatory protein(COR) protein expression, without cold acclimation of plants have certain resistance. Therefore through transforming the CBF genes can improve the frost resistance of tobacco, to improve tobacco yield and improve its quality. When the plant is in a state of low temperature stress, low temperature dehydration plant and plant cell membranes will cause severe damage, and anti-cold cell membrane structure for the protection of the transcription factor plays an important role in bringing the plant to slow to some extent or boycott hypothermia.In this experiment, sterile tobacco leaf receptor material, the use of Agrobacterium-mediated transformation of tobacco leaf disk method. The main conclusions of this paper are as follows:1. Tobacco(tobacco 100) leaves were used as explants, the optimum medium by screening the various stages of the establishment of regeneration system of tobacco. Among them, the medium MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L was the best medium tobacco leaves adventitious buds differentiation; MS + 6-BA 2.0 mg/L is the best tobacco culture of adventitious bud proliferation group; MS + NAA 0.1 mg/L is the best tobacco the rooting culture medium.2. The tobacco leaves into Agrobacterium after the screening medium containing different concentrations of Kan screened resistant buds resistance threshold of Kan’s. This threshold is at a concentration of 50 mg/L Kan solution.3. Establish the optimal transformation system of tobacco: explants pre-cultured 48 h, with OD600 of about 0.4 Agrobacterium suspension infected explants 5 min, inoculated with acetosyringone(AS) co-culture culture groups, co-cultured for 48 h sterilized.4. Tobacco leaf discs by genetic transformation, 5 numbers of transgenic plants. CTAB method using transgenic DNA and PCR testing, approximately 750 bp to give a clear band indicating CBF1 gene was successfully transferred into tobacco.5. Transgenic tobacco physiological indexes detected after 24 h treatment at 4 ℃and 22 ℃recovery after treatment 72 h, SOD, POD, free proline, CAT activity were increased and higher than untransformed tobacco activity, MDA activity decreased and is always lower than the tobacco gene transfer activity was not described to a certain extent, improve the cold resistance of plants.