| Cold is a major constraint of crop productivity, which produces almost two hundred billion US dollar' lost in the world per year. Plant cold tolerance is regulated by many genes, and only in the case of some cold-related genes are transcriptionally activated at the same time, plant can survive under cold stress. Recently, scientists focus their research on cold-related transcription factors, and several CBF (C-repeat Binding factor) genes from different plants were cloned and characterized. CBF transcription factors can activate expression of many cold regulated (COR) protein by binding to the CRT/DRE DNA element that locates in the promoter of COR genes, which significantly improve plant's freezing tolerance ability. In this paper, a novel CBF transcription factor from Malus baccata (MbCBF) was cloned (GenBank accession number: EF582842.). The plant expression vector of MbCBF was constructed and transferred to Arabidopsis thaliana. The main results are as follows:1. A pair of degenerate primers was designed by comparing CBF genes from plants which are published in Genebank, and the conserved region with a length of 461 bp of the transcription factor CBF gene by both of the reverse transcription polymerase chain reaction (RT-PCR) and PCR from M. baccata. Then, two pairs of specific primers from the conserved fragment of targeted gene were designed to isolate 5' and 3'ends of CBF gene by inverse PCR (iPCR). One fragment of 1814 bp is amplified by iPCR. After being spliced, a predicted full length of CBF gene with 2284 bp from M. baccata was obtained, and an entire open reading frame (OR.F) of 251 anino acids was detected with a potential start codon at 790 bp and a stop codon at 1545 bp from upstream. In order to ensure if there are any introns in the coding domain of CBF gene, a pair of primer was designed according to predicted full length coding domain of CBF gene and PCR was done by using gemomic DNA as template. And the result showed that there is no intron in CBF gene because both of the genomic sequence and the coding domain of CBF are exactly same. Furthermore, homology was compared of MbCBF with CBFs from other plants by using DNAman software. The result indicated that MbCBF is 66.93% homologous with Prunus avium and 55% with Hevea brasiliensis, which demonstrates MbCBF belongs to the plant CBF gene family. 2. The results of structure analyses of MbCBF:1) MbCBF shares high homology of both nucleotide and animo acid sequences with other CBF genes from plants.2) MbCBF gene encodes an acidic protein with pI value 5,01.The predicted molecular mass is 27.9KD.3) MbCBF sequence contains a functional domain:AP2 binding domain, on the both sides of which there exist two short peptides: PKK/RPAGRxKFxETRHP and DSAWR.4) The MbCBF sequence contains acidic activation domain. But there is no NLS (Nuclear Localization Signal), signal peptide and transmembrane domain.5) Animo acid sequence alignment of MbCBF showed that it contains conserved Alpha helix and Beta turn domain.6) It was possible to construct a partial 3D-structure of MbCBF by homology modeling.7) Phylogenetic tree of MbCBF and other CBF genes from 12 plants suggest that MbCBF and sweetcherry share the common origin.3. Plant expression vector construction of MbCBF was done. The expression vector was directed by 35S promoter, GUS reporter gene was replaced by MbCBF in the pBI121 plasmid at BarnHâ… and Sacâ… restriction site. The new constructed plant expression vector is called pBC35S.4. pBC35S construct was then transfered to Arabidopsis by Floral Dip method. And the transformed Arabidopsis lines were obtained, which will be used for further gene functional analysis.
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