Cloning, Expression Of A Novel CBF Transcription Factor Gene (OsCBF1) In Rice And Expression Vector Construction Of OsCBF1

In northern part of China, the abiotic stresses such as low temperature, salt anddrought are frequently occurance during crop production. It has an important role toenhance the crop productivity by increasing the biotic tolerance in crops. It is found thatthere exists the signal transduction and programmed expression of some genes involvedinto the tolerance of the stresses when plants are growing at the abiotic stess condition.Among the signal transduction components, the transcription factor CBFs are importantand have important roles to improve the tolerant abilities of above stresses in crops.In this study, a novel rice transcription factor gene OsCBF1 classified into CBF groupwas cloned. The expression pattern of OsCBF1 was analyzed by semi-quantitive RT-PCR.Meanwhile, a binary expression vector fused the open reading frame (ORF) of OsCBF1was constructed. This provides a basis for further analysis of the gene function and themechanism of expression regulation.The main results of this study are as follows:1. By BLAST analysis in international bioinformatics website NCBI, a putative CBFtranscription factor gene (GeneBank accession number: NP₉10360) which has not beencharacterized was identified. It was named OsCBF1 in this study. The full length cDNA ofOsCBF1 was PCR amplified by using the genome DNA of rice cultivar TP309.2. The full legth of OsCBF1 is 772 bp, including an open reading frame (ORF) 645 bpand 5' and 3' untranslated region of 27 bp and 100 bp, respectively. There are no introns inOsCBF1. The OsCBF1 encodes 214 amino acids and the molecular weight is 23.5 KD. Anuclear location signal (NLS), a DNA binding domain and a transcription activating motifcould be identified in OsCBF1.3. Under normal growth condition, low temperature, salt and drought treatments, theexpression patterns of OsCBF1 and other eight putative rice CBF genes homozegous toOsCBF1 were evaluated. The transcripts of OsCBF1, CBF1-like, RCBF3, unnamed1 andunnamed 6 were identified. But others were not, maybe due to no transcripts or lower thanthe detected level of expremental method, or related to unsuitable primer design for thesegenes.4. The expression of OsCBF1 showed root specific. The transcripts of CBF1-like,unnamed1 and unnamed6 were increased under low temperature. 2 h treatment of low temperature dramatically increased the transcripts of above genes. Up to 4 h, theexpression level was decreased in CBF-like and gradually increased in unnamed6compared to those in 2 h, repectively. The expression of unnamed1 was induced by lowtemperature with no transcripts checked under normal growth condition. It is suggestedthat OsCBF1, unnamed1 and unnamed6 have possibly important roles in mediating thesignal transductions of low temperature in rice.5. After 3 h treatment of salt, the transcripts of OsCBF1, CBF1-like, RCBF3 andunnamed6 in roots were increased compared to those in CK, with much more increases inOsCBF1 and unnamed6 among them. These results imply that above genes arc involved inthe salt signal transduction in root. There were different expression patterns of above genesin leaf, showing no expression of OsCBF1, induction of CBF1-like, low regulation effectof RCBF3, and down-regulated of unnamed6. Therefore, the capabilities and mechanismsof above CBF genes mediating salt signals are not same.6. Three hours of drought treatment have no effects on the expression levels ofOsCBF1, CBF1-like, RCBF3, unnamed1 and unnamed6 in root. The leaf transcripts ofOsCBF1, CBF1-like and unnamed were affected little by drought. But the expressionlevels were obviously down-regulated in RCBF3 and unnamed6. This indicated thatRCBF3 and unnamed6 are involved in drought signal transductions in leaf.7. By using DNA recombinant technology, the binary expression vectorpCAMBIA1305-OsCBF1 which fused the open reading frame (ORF) of OsCBF1 wasconstructed. A positive clone of Agrobacterium tumeficiens (Strain LBA4404) harbouringthis binary vector was isolated. This work has provided a basis for identifying the functionof OsCBF1.8. The 5' flanking region of OsCBF1 was PCR amplified as the TP309 genome DNAto be the template. It is found that this promoter region contained some importantregulatory elements involving the regulation of downstream genes. Based on the moleculartechniques, the binary expression vector pCAMBIA1305-OsCBF1P fused the promoterregion was constructed. This will be useful to identity the function of this promoter andfine-regulatory elements regulating the OsCBF1 expression in future.