Isolation And Functional Analysis Of A New Cold Induced CBF Gene From Camellia Sinensis

Tea (Camellia sinensis) is a perennial evergreen woody plants which is one of the most important economic crops in China. Tea industry plays an important role in the social and economic development of our country, recently. The growth of the tea which prefer to warm condition has requirement of temperature that is between10℃and35℃so that the tea can grow up regularly. The root system will stop growing under8℃. Freezing injury will occur when the temperature is lower than the limit temperature that the tea can endure, it can also effect growth and development of the tea. CBF transcription factor is the member of ERF subfamily in AP2/ERF gene family which plays an important role in plant response to low temperature stress.We obtained a cold-induced cDNA sequence of CBF named Cs-COR041(GenBank ID FE942095) in the tea. The gene-specific primers of CsCBF3are designed on the basis of CsCOR041sequence with SMART cDNA library as a template. Then we obtained CsCBF3gene3’end(about450bp) and5’end(about600bp). To get the full-length of CsCBF3(1162bp),(GenBank ID KC702795), we splice the cDNA sequence which we known and the gene3’end (about450bp) and5’end which we obtained. This sequence includes a complete open reading frame (720bp) coding a polypeptide which composes of239amino acid residues. The theoretical molecular weight of the protein coded by this nucleotide is26.44kDa and the isoelectric point is5.22. After analyz the system evolution of CsCBF3, the result suggests that the tea CsCBF3has a close relationship with aspen and grape. The amino acid sequence alignment analysis showed that CsCBF3has a conserved AP2domain. The Hidden horse model of AP2domain coded by each species CBF gene shows that CsCBF3AP2domain is highly conserved except for several sequence.The analysis of transcriptional activity of CsCBF3were performed by the yeast one-hybrid experiment, and the result suggest that the CsCBF3protein has transcriptional activation activity and specifically combined with DRE/CRT element. Further investigation discovered that, the outside regions of CsCBF3protein activation domain has inhibiting effect on the activation function.。The CsCBFl and CsCBF3genes were constructed to the plant expression vector pCABIA1300S vector respectively, which has a35S strong driving promoter and a site of Hygromycin resistance selection marker. The CsCBF1-pCABIA1300S and CsCBF3-pCABIA1300S vectors were constructed successfully. Transgenic tobacco were obtained by the Agrobacterium-mediated transformation and hygromycin selection. At last,23plants of CsCBFI and18plants of CsCBF3To generation transgenic tobacco were obtained respectively by PCR identify method.The pCzn1-CBF1and pCzn1-CBF3protein expression vector were constructed. The recombinant plasmid was transformed into Escherichia coli Arctic Express. Induced by IPTQ the recombinant protein existed in the form of inclusion body. To separate the inclusion body, we need denaturation and renaturation the protein, then by the Ni affinity chromatography; we get the recombinant protein of CBF3and CBF1fininally.