Cloning And Transformation Of BpDREB Gene Of Transcription Factor In Chinese Cabbage

The research found that the tolerance of plants have been related with two kinds of genes. One is the structural gene that translated into tolerance-related protein; the other is transcription factor gene that regulate the signal transduction pathway and the expression of structural gene, such as bZIP transcription factor,MYC transcription factor, DREB transcription factor. Over the years, the study of the first category gene on the structural gene expression characteristics、the structure and function of gene research has been a great success. But the researchers still know less about the downstream signal transduction pathway and further gene expression under environmental stresses such as freezing, drought, and salt. Therefore, the study that focus on the clone of the transcription factor is a hot pot in plant resistant molecular biology.In plants, there are many transcriptional factors. And the dehydration responsive element binding (DREB) element play an important role on plant resistance to adversity, for example dehydration, high salt and low temperature. DREB proteins belong to AP2/EREBP family and they are specific exists in plants. This kind of transcription factor all contain AP2/EREBP DNA domain, and can specially bind to DRE cis-acting element. The core sequence of the DRE cis-acting element is CCGAC. The research indicated that the tolerant structural gene all contain DRE core sequence in many kinds of plants. The over expression of DREB can regulate the expression of series of genes at the same time. Consequently, the whole tolerant of the plant cells were improved. So first we clone a transcription factor and then transform it into the plant, it is an effective way to improve the plant tolerance.In this experiment, we look up a series of DREB related genes on net that bank in GeneBank, DDBJ etc. Then design a pair of probes base on these sequences. The third, we clone a DREB gene from DNA in Chinese cabbage Qiulv60. It was composed of 777 base pairs; the opening reading frame is from the sixth base pair to the776 base pair; it can be translated into a polypeptide that was composed of 256 amio acid. It also contained a conserved AP2/EREBP DNA domain, and V14 and E19 that were conserved in DREB subfamily. So it was named BpDREB(Brasssica pekinensis DREB).With the same primers, we also got the same gene from the cDNA of the Chinese cabbage leaves which was treated 12 hours under 4℃. By the analysis, we confirmed that the two genes were the same. From this point, we can conclud that the gene BpDREB is functional.In order to study the function of this gene, we construct a transformed vector pCAMBIA1301-BpDREB with expression vector pCAMBIA1301. Then it was transformed into the Agrobacterium tumefaciems to get a posite clone, at last it was transformed into the Arabidopsis thaliana by the flore dip method. After three weeks, many seeds can be collected. In order to get a transgene plant, first, the seeds were cultured on the Kanamicine medium. During this procedure, many seeds die except the transformed plant. About 10 days later, the survived plant can be transformed into the normal enviroment. At last, we need to do some molecular biology detect to ensure the transformed plant and its resistance. This research supply a reliable proves on the functional research of the plant resistance.