Study On Transformation Of Tomato With ICE1 Gene From Arabidopsis

Cold is an environmental factor that limits the geographical distribution and growing season of many plant species, and it often adversely affects crop quality and productivity. Cold stress causes to the worlds lose $2000 billions every year. Study the transcriptional factor that regular cold gene play a significant role. CBF (CRT/DRE binding factor) genes were shown to be key role that control the expression of cold-regulated genes. ICE1(inducer of CBF expression 1) is the only transcription factor that has been identified to act directly on the CBF promoter, ICE1 is a master switch that controls CBF gene. ICE1 is an upstream transcription factor that positively regulates CBF genes expression in the cold. Although ICE1 is constitutively expressed, it activates CBF gene expression only upon cold treatment. Over expression of ICE1 cause to trigger the CBF genes expression, then CBF bind to CRT/DRE and activates expression of the downstream COR gene, which confer chilling and freezing tolerance to plants.ICE1 may regulate cold responsive gene expression in other way. But up to now, we know little how it regulates those genes. In this study, we transfer of cold-regulated ICE1 gene into tomato mediated by Agrobacterium tumefaciens. The main results of the experiment as follows:(1) Total RNA was abstracted from Arabidopsis, then after RT-PCR reaction, Clone and DNA sequence analysis, plant expression vector was constructed which ICE1 gene was inserted into it.(2) The explants were cotyledon, culture medium supplied with different hormone combination. This was the best bud induction medium: MS +1mg/L ZT+1 mg/L IAA+3% sucrose+0.8% Agarose,PH5.8.(3) Study the factors that effect tomato transformation. An efficient gene transformation system was established, and the optimum conditions for the transformation as follows: Cotyledons were pre-culture 2 days on bud induction medium; Agrobacterium were diluted, in which explants were soaked for 20 minutes with another 100μmol/L AS. Then the explants were co-cultured for 3d. After that, the explants were cultured on select medium. When shoots were more than 1cm high, they were cut and transferred on medium (1/2MS+0.1mg/L IAA+15mg/L Hyg+300 mg/L Cef) for rooting.(4) After resistance selection, differentiation and rooting culture, we got transgenic plants. With the plasmid DNA as positive control and wild tomato as negative control, the PCR result showed that 5 regenerated pants of them had amplified idea DNA band, which had the same location as amplification band of the positive plasmid. The results proved that ICE1 gene have been integrated into tomato genome.