Genes And Their Expressin In CBF Cold-response Pathway In Chinese Cabbage

CBF genes are at nodes of regulatory network in Arabidopsis response to cold stress. They act to integrate the activation of multiple components of the cold acclimation response. Though existing many cold-response pathway in plant, the CBF pathway have been studied extentively, and its cold-resisting function was confirmed firmly. So the genes in it are importment resources for plant cold-resisting genetic engineering. In our work, several genes in the CBF cold response pathway were isolated by PCR from Chinese cabbage treated with low temperature. Sequence analysis and function prediction were carried out with different biological program. Gene expression pattern and transcription regulation under various treatments were investigated. The summary is as follows.1. A coding sequence of COR-like gene was cloned with degenerate primers from Chinese cabbage (B. pekinensis) , named BcCOR. The gene encodes a deduced protein of 129 amino acids. Hydrophilicity prediction shows BcCOR is a highly hydrophilic small protein and it possesses a transporting peptide sequence locating the protein into chloroplast where BcCOR functions. The secondary structure analysis shows BcCOR protein is rich in alpha helix (72.87%) and random coil (17.05%) . These structural characteristics of BcCOR are similar to that of AtCOR15a from Arabidopsis. In addition, BcCOR shares a 80.2% nucleotide identity and a 71.4% amino acid identity with AtCOR15a. All of these indicate that BcCOR is the homologus gene of AtCOR15a and may be involved in cold-response process in Chinese cabbage as AtCOR15a in Arabidopsis.2. The RNA blotting results show that both cold and dehydration treatments can induce the expression of BcCOR as the same pattern. In addition, the patterns of transcription profiling are approximately similar to those of AtCOR in Arabidopsis. This indicates that the genes from the same family have the similar transcription profiling and conservative function. Nevertheless, the transcription expression of BcCOR is different from that of AtCOR in some ways. On the one hand, The BcCOR transcription seems more sensitive to cold than to dehydration treatment. On the other hand, unlike AtCOR in Arabidopsis, ABA treatment can't induce the mRNAaccumulation of BcCOR. The difference between expression patterns of homologous genes suggests existing a functional diversity of these genes. We propose that the diversity is caused by the difference of regulatory reign of these genes.3. Two homologous genes of the Arabidopsis C-Repeat/Dehydration-Responsive Element Binding Factors (CBF/DREB1) transcriptional activator were isolated by PCR from Chinese cabbage (B. pekinensis cv. Qinbai5 ) and designated as BcCBFl and BcCBF2. Each of them encodes a putative CBF/DREB1 protein with an AP2 DNA-binding domain, a putative nuclear localization signal and a possible acidic activation domain. Deduced amino acid sequences show that BcCBFl is very similar to the Arabidopsis CBF1, whereas BcCBF2 is different in that it contains two extra regions of 24 and 20 amino acids in the acidic domain.4. The mRNA accumulation profiles made clear that BcCBFl and BcCBF2 expression was strongly induced by cold treatment, but did not response to dehydration and ABA treatment similarly. However, the cold-induced accumulation of BcCBF2 mRNA was rapid but short-lived compared with that of BcCBFl.5. The levels of both BcCBFs mRNA were higher in leaves than in roots when plant was exposed to cold, whereas, salt stress caused higher accumulation of the BcCBF2 mRNA in roots than in leaves, suggesting that organ specificity of BcCBFs gene expression probably lie on different stress.6. The mRNA accumulation of BcCBFl and BcCBF2 was greatly enhanced by light compared to darkness when seeding was exposed to cold. It was concluded that the two protein BcCBFs may be involved in the process of plant response to cold stress through an ABA-independent pathway and that there also exists a crosstalk between light signaling conduction pathway and the cold response pathway in B. pekinensis similar to Arabidopsis.7. The comparison of gene expression patterns suggests the vertical regulatory relation between BcCBFs and BcCOR initially. Combination of Agroinfiltration technique and Semi-quantitative PCR analysis, the researches of transient gene expression further confirm that BcCOR is regulated by BcCBFs in Chinese cabbage. These results indicate that there exists a CBF cold-response pathway in Chinese cabbage as in Arabidopsisand that it plays a crucial and conservative role in the anti-freezing molecular mechanism in plants.In conclusion, the CBF cold-response pathway was also found in Chinese cabbage, a cold- endurance crop. This indicates that there is a conservative machenism in plant cold-resisting process and that the genes cloned in this research have potential cold-resisting function. The different expression profiles between homologous genes suggest the diversity and complexity of their functions. The studies also suggest there exsit extensive crosstalks between CBF and other pathways.