Cloning, Subcellular Localization And Functional Analysis Of CBF、COR Gene In Brassica Campestris L.

Low temperature, drought and salt stresses are disadvantageous for the plantsgrowth and development. Recently, scientists focus their research on transcriptionfactors, and several CBF genes from different plants were cloned and characterized.Transcription factor CBF can bind specific core sequence of CRT/DRE DNA elementA/GCCGAC that locates in the promoter of COR genes, and activate expression ofmany COR protein, can induce the downstream functional genes which have responseto salt, drought and low temperature. Based on the importance of CBF and COR insignaling, this dissertation analyzed of CBF and COR gene, which in Longyou6andTianyou2. We did a preliminary study about two kinds of Brassica campestris L.genes sequence features, expression models, subcelluar localization. The main resultsare as follows:1. We isolated a new transcription factor CBF and a new cold-regulated geneCOR from Brassica campestris L. Longyou6. The cloned full-length cDNA of CBFwas1025bp with an open reading frame (ORF) of645bp comprising215amino acidresidues with the predicted molecular mass of23.8kD and with a PI of4.86. CORbioinformatic analysis reveals that the cDNA is641bp in length with an openreading frame of390bp which encodes a poly-peptide of129amino acids. Themolecular weight of the predicted protein is13.9kDa, with the calculated isoelectricpoint (PI) being5.2. Senior predict protein structure show that CBF rich in irregularcurly and alpha helix, COR mainly alpha helix, both for the hydrophilic protein, nosignal peptide sequence.2. CBF, COR expression patterns under abiotic stresses were analyzed byReal-time quantitative RT-PCR. CBF, COR were expressed in the stems, leaves, andhypocotyls of the both kinds of Brassica campestris L., with almost no tissuespecificity. Low temperature, high salt, ABA, H2O2stress can can induce theexpression of two rape CBF, COR genes, and rapeseed Longyou6CBF, CORtranscription level was significantly higher in Tianyou2. CBF, COR genes inLongyou6against abiotic stress play a role in the process. MAPKK inhibitor U0126pretreatment12h before the above four kinds of adversity stress, found two rape CBF,COR genes can produce a certain amount of response in Longyou6CBF, COR transcription level was significantly higher in Tianyou2, but the expression level ofboth are significantly decreased, Show that the MAPKKK-MAPKK-MAP kinase ofMAPK signal pathway and composed of CBF, COR response interaction betweentranscriptional network in low temperature.3. The fusion protein pBI121-CBF-GFP、pBI121-COR-GFP was expressedtransiently in onion epidermis. The Fluorescence microscopic examination showedthat the pBI121-CBF-GFP fusion protein was targeted into the cytoplasm,pBI121-COR-GFP fusion protein was targeted into the nuclear. The result clearlyindicates that CBF is localized in the cytoplasm, and COR is localized in the nucleus.