Cloning Of DREB-like Transcription Factor CDNA In Chinese Cabbage And Construction Of Plant Expression Vector

Drought, salinity, low-temperature and other abiotic adversity stress, impacting on plant growth development significantly, are the direct cause of reduction in crop production. Increasing the stress resistance of plants is important in agricultural production, improvement of ecological environment and sustainable development, because it can reduce the stress injury, increase crop production and save fresh water resources. The DREB transcription factor can specifically combine with the DRE (dehydration responsion element) and activate expression of many stress-induced genes , resulting in regulating and controlling the molecules response to drought, hyperhaline and low-temperature stress. The DREB transcription factor, regulating the expression of functional genes under the stress and increasing the capacity of resistance and endurance to adversity of plant, is the best adversity resistance gene in plant by far.In this study, Chinese cabbage dealt with the adversity was selected as experiment material. Using RT-PCR technology, we obtained the cDNA sequences of Chinese cabbage DREB transcription factor's. Under adversity stress, stress-induced expression patterns of DREB transcription factor in Chinese cabbage was studied and plant expression vector was constructed successfully. The results are as follows:1. According to published gene sequence of Brassica napus DREB transcription factor, primers DR2-1 and DR2-2 were designed and two genes with 645bp in length were amplified by RT-PCR. Each gene contained a complete sequence encoding 214 amino acids, named BcDREB1 (GenBank accession number: EU924266) and BcDREB2 (GenBank accession number: GQ122331) respectively. Homological analysis showed that over 90% amino acids in the two sequences were similar to Brassica napus DREB2-2 gene and Brassica juncea Coss DREB1B gene.2. SMART and Protparam analysis indicated that the deduced relative molecular weight of the amino acid was 23.90KD for BcDREB1 and 23.85KD for BcDREB2, and the isoelectric point was 4.88 BcDREB1 and 5.21 for BcDREB2. Furthermore, a typical AP2 structure domain was found in both of gene structures.3. Using semi-quantitative RT-PCR. analysis of expression modes showed that BcDREB gene expression induced by low temperature, drought and hyperhalin. The gene started to express after 1h and reached the highest level after 3h under the low-temperature stress, started to express after 1h and reached the highest level after 12h under the drought stress, and started to express after 3h and reached highest level after 12h under the high-salt stress.4. The sensed specific primer DR-F and DR-R,containing XbaI and BamHI restriction sites, were designed.Plasmid containing BcDREB1 gene and pCAMBIA2301 vector was digested with BamHI and XbaI,respectively. The T4 DNA Ligase was used to be ligated, sensed plant expression vector pCAM-BcDREB1 of BcDREB1 gene was built in Chinese cabbage.5. The antisensed specific primer DR-F and DR-R were designed which contained BamHI and XbaI restriction sites. Plasmid containing gene BcDREB1, BcDREB2 and pCAMBIA2301 vector were digested with BamHI and XbaI, respectively, connected using T4 DNA Ligase, antisense plant expression vector anti-pCAM-BcDREB1 and anti-pCAM-BcDREB2 of BcDREB1, BcDREB2 gene were built in Chinese cabbage.6. The plant expression vectors pCAM-BcDREB1, anti-pCAM-BcDREB1 and anti-pCAM-BcDREB2 were transformed into Agrobacterium EHA105 using freeze-thaw method. The PCR amplification specific primers was further carried out in transformed bacteria, expression vectors were confirmed to transfer into the Agrobacterium.