Cloning And Primary Function Analysis Of CBF/DREB1 Homologous Genes In Prunus Mume

Mei (Prunus mume), a native plant in China, is important for its beautiful flower and edible fruit. The growth region of Mei in north China is mainly restricted by the early coming, long-standing low temperature and the cold-dry wind. The primary objectives for Mei flower researchers are that to breed the strong cold-resistant cultivars for a long time. Using the traditional breeding methods, some high cold-resistant cultivars were identified. However, the traditional breeding programme should take a long time for tree improvement. The application of transgenetic technology in cold tolerance breeding of Mei flower will overcome the restrictions.CBF/DREB1 homolog genes are ubiquitous in cold acclimated plants. Their expression can be induced by low temperature stress. Up-regulated expression of these genes will promote the cold tolerance and some other stress resistances for plants. CBF/DREB1 homolog genes from different species showed conserved gene structure and regulatory role of stress-induced genes that belong to the downstream pathways. Thus, the rapid development of molecular breeding, in combination with the key genes, brings about new hope for cold-resistant cultivated programme of Mei flower.This work based on the former researchs of the CBF/DREB1 transcription factor. Molecular cloning, expression analysis of CBF/DREB1 homolog genes, measure and comparison of the physiological indices of the transgenic tobacco under cold stress were done. The main results are as follows:1. We combined RT-PCR and RACE strategies to clone the CBF/DREB1 homolog genes from Mei flower. Two full length genes, namely PmCBFa and PmCBFb and another partial gene segment PmCBFc were obtained from the Prunus mume ’Xue Mei’ (GenBank accession numbers:HM099909, HM099910, HM099911). Two gene segments PmbCBFa and PmbCBFb were also cloned from another cultivar P. mume var. Bungo (GenBank accession numbers:HM099907, HM099908). PmCBFa and PmCBFb were arranged in tandem on a 3.7kb genomic sequence.Using this genomic segment, promoter prediction was performed on BDGP website (http://www.fruitfly.org/seq_tools/promoter.htm) and PLACE database (http://www.dna.affrc.go.jp/htdocs/PLACE). The result showed that multiple promoter structure characters were present in this genomic segment. The nucleotide and deduced amino acid sequences of PmCBF genes were aligned with other CBF/DREB1 orthologous sequences. Comparison of the various sequences showed high identities among them. It also revealed that there were several single nucleotide site differentiations in PmCBF genes between different Mei flower cultivar. Phylogenetic analysis placed the putative PmCBF proteins in the CBF/DREB1-subfamily of the dicots.2. Expression of PmCBF/DREB1 genes in leaves of Prunus mume on simulating cold, drought stress and mechanical agitation was detected through semi-quantitative RT-PCR. The results showed that PmCBF/DREB1 genes could be reduced by low temperature or drought, or by mechanical agitation, and the variation trends of the gene expression is similar to other CBF/DREBl homologs that had been reported from other plants.3. Through double digestion and plasmid recombination, an over-expression vector, PmCBFb2300-cis, was obtained containing PmCBFb complete CDS. The transformation plant of tobacco was got via Agrobacterium tumefaciens. Some physiological indexes, such as SOD activity, MDA content, soluble sugar content, and chlorophyll content of transgenic tobacco plants, were determined. Statistical analysis showed transgenic tobacco has a slightly higher tolerance to cold, mainly embodying in the variation range of the highest content of toxic substances during clod hardening, such as MDA, and decrease of content of osmotic regulation substances and cleanout of toxic substances after cold acclimation.This subject is just the first report of the molecular cloning of cold resistance genes in Mei flower, but some efforts must be done on function and regulationary nets of the critical transcription factor of cold tolerance in blossom stage of Prunus mume.