Cloning Of Gene Encoding CBF Transcription Factor From Rosa Chinensis (RcCBF) And Transformation Studies Of RcCBF In Tobacco

Plants are exposed to environmental stresses such as low temperature, drought and high salt, which causeadverse effects on the growth and development of plants. CBF(C-repeat binding factor) is a kind oftranscription factors that conferred to the tolerance of plant abiotic stresses. These proteins are able to bindto the CRT/DRE (C-repeat/dehydration responsive element) DNA element in the promoter of some cold,drought and high salt-related genes, and transcriptionally activated these genes' expression. Therefore, itwould be a good strategy to improve plants' tolerance to abiotic stresses by transforming CBF genes. In thispaper, a novel CBF transcription factor from Rosa chinensis (RcCBF) was cloned (GenBank accessionnumber: EF583559). The plant expression vector of RcCBF was constructed and transferred to tobacco byAgrobacterium tumefaciens-mediated leaf discs transformation. The main results are asfollow:1. A pair of degenerate primers was designed by comparing DREB genes of several plants which arepublished in Genebank. The degererate primers were used to isolate the conserved region with a length of365 bp of the transcription factor CBF gene from Rosa chinensis by polymerase chain reactin (PCR).2. Two pairs of specific primers from the conserved fragment of targeted gene were designed to isolate5' and 3'ends of CBF gene by inverse PCR (iPCR). One fragment of 1400 bp is amplified by iPCR. Afterbeing spliced, a predicted full length CBF gene of 603 bp from R. chinensis (RcCBF) was obtained.3. From the predicted full length sequence of RcCBF gene, a specific primer was designed to isolatethe genomic structure of poplar DREB gene and the results showed that RcCBF doesn't have intron incoding domain.4. By DNAMAN software, homologous alignment was done of RcCBF gene with other genes fromCBF gene family published in Genbank, the result showed that RcCBF has the conserved AP2 region asone of the characteristics of DREB gene family, which demonstrates we have cloned the transcription factorCBF gene from R. chinensis successfully.5. Construction of the overexpression vector of RcCBF was done. The expression vector was drivenby a 35S promoter, the GUS gene was replaced by RcCBF of pBI121 plasmid at BamHâ… andSacâ… restriction site, respectively. The new constructed plant expression vector is called pBI-CBF.6. pBI-CBF construct was then transfered to tobacco by Agrobacteriumm tumefaciens-mediated leaf discs transformation. PCR results of transgenic tobacco demonstrated the transformation was successful.