Analysis Of Mechanism In Response To Drought And Cold Stresses And Cloning Of The Key Cold-related Gene BrCBF1 In Brassica Campestris

Cold and drought stresses are profoundly the most important environment facts affecting crop growth and development and leading to decrease crop yield. Therefore, it is important for studying the mechanisms of plants in response to drought and cold stresses. We studied the tolerance to low temperature and drought stress of three cultivars of Brassica chinensis, which are BcL.1, BcL.2, BcL.3. The results are as follows:1. BcL.3 showed enhanced tolerance to low temperature stress compared with BcL.1, BcL.2, as indicated by higher survival rate (2-3 times) and significantly lower electrolyte leakage than that of BcL.1, BcL.2. The plant damage caused by low temperature stress of BcL.3 is lower than that of the other two cultivars.2. BcL.3 also displayed enhanced tolerance to drought stress compared with BcL.1, BcL.2, as indicated by higher survival rate (2-3 times) and a lower water loss of detached plant than that of BcL.1, BcL.2.3. In order to study the mechanism of BcL3 in regulation of low temperature and drought tolerance,we detected soluble sugar content of the three cultivars. The soluble sugar content of BcL.1, BcL.2, BcL.3 were increased but there was no significant difference after low temperature and drought treatment, suggesting that low temperature and drought tolerance of BcL.3 were not related to soluble sugar accumulation.4. Proline content of the three cultivars all increased after drought treatment. The content of proline in BcL.3 was significantly higher than that of BcL.1, BcL.2. But the proline content of three cultivars have no significant difference after low temperature stress,suggesting that the tolerance of BcL.3 to drought stress may caused by altering the proline content.5. To further validate the cold and drought resistance mechanism of BcL.1, BcL.2, BcL.3, we detected the expression of proline metabolism related genes, the expression level of proline biosynthetic gene P5CS in BcL.3 was sgnificantly increased after drought treatment, the PDH gene expression of proline degradation pathway in BcL.1, BcL.2, BcL.3 showed no significant difference, that suggesting P5CS gene highly expressed increased proline content in BcL.3 under the drought stress. We conclude the same support evidence after the further detection of P5CS enzyme activity. However, the transcription level and enzyme activity of P5CS and PDH had no significant difference in the three cultivars after low temperature treatment.6. In order to further study the reason of low temperature tolerance of BcL.3 respect to BcL.1, BcL.2, we detected related gene expression in CBF/DREB1-dependent signaling pathway under low temperature stress. The results showed that CBFI,a. key gene in CBF/DREB1 pathway, is highly expressed under the regulation of the upstream factor LOS4 and its own regulate factor CBF2 under low temperature stress in BcL.3, enhanced tolerance to low temperature stress compared with BcL.1, BcL.2 by activating the expression of downstream gene COR 15A.7. The full-length BrCBF1 coding sequence was amplified from cDNA of BcL.3 and cloned into the pART27 vector. The resulting construct was transformed into Agrobacterium strain GV3101 for transformation into the Col-0 plants by the floral dip method. The transgenic lines were isolated by antibiotic screening and genetic identification.