Cloning And Characterization Of Cold-Regulated Genes In Brassica Campestris Ssp.chinensis

Many plants have an in-built ability to adapt the low temperature, and this is coldacclimation. The expression of cold-related genes is also modulated by cold acclimation.Analyzing amino acid sequence and identifying the function revealed that cold acclimationwere controlled by several main genes and modifying genes. In this study, several geneswere cloned from Brassica campestris ssp. chinensis by rapid amplification of cDNA ends(RACE): a novel cold-regulated gene BrCOR14 and its up-stream transcriptional activatorBrCBF and BrCBF's up-stream transcriptional factor BrICE and a regulator of other coldresponsive gene BrLOS2.1. Cloning and characterization of cold responsive gene BrCOR14.By RT-PCR and RACE strategy with gene specific primers designed from Arabidopsisthaliana cold-responsive genes, the full-length cDNA gene named BrCOR14 (Genbankaccession number DQ192529) was cloned from the leaves of Brassica campestris ssp.chinensis which were cold-induced for 7 days. The full-length cDNA of BrCOR14 was549bp and contained a 387bp opening-reading frame(ORF) encoding 13.749kD peptideincluding 128 amino acids with pâ… of 5.61. Further comparison to Brassica napusCOR14,BN115,BN19 genes showed the identity were 96.9ï¼…, 78.87ï¼…and 76.76ï¼…,respectively, and the similarity to Arabidopsis COR15a and COR15b was above 60ï¼…. Thepredicted BrCOR14 protein was found to have a similar function to cor15 and LEA. Thesecondary structure analysis revealed that BrCOR14 strongly resembled other cor genes.The result of semi-quantitative RT-PCR demonstrated that expression of BrCOR14 genewas induced by low temperature. All results presented implied that BrCOR14 might havethe similar function possessed by other COR genes in increasing plants' freezing tolerance.2. Cloning and sequence analysis of BrCBF, an up-stream transcriptionaland activated factor BrCOR14.BrCOR14's up-stream transcriptional factor BrCBF (Genbank accession numberDQ402470) was cloned from the leaves of Brassica campestris ssp. chinensis which werecold-induced for 12 hours. The full-length cDNA of BrCBF was 1003bp and contained a648bp opening-reading frame (ORF) encoding 24.05kD peptide containing 216 aminoacids with pâ… of 5.32. Further comparison to Brassica napus and Brassica juncea genesshowed the similarity were 84.26ï¼…and 65.61ï¼…, respectively. The similarity to Arabidopsis thaliana and Capsella bursa-pastoris was above 70ï¼…. The predicted BrCBF protein wasfound to have a potential AP2 DNA-binding motif. Bioinformatics analysis revealed thatBrCBF strongly resembled other CBF genes. Semi-quantitative RT-PCR demonstrated thatthe expression of BrCBF gene was induced by low temperature.3. Cloning and sequence analysis of BrICE, an up-stream transcriptionaland activated factor BrCBF.The BrICE was cloned using RT-PCR with cDNA isolated from Brassica campestrisssp.chinensis, which was cold-induced for eight hours. The cDNA of BrICE was 1558bpand contained a 1491bp opening-reading frame (ORF) encoding 497 amino acids. Thefurther comparison showed that BrICE identity to Arabidopsis thaliana ICE1 and Capsellabursa-pastoris CbICE53 were 87.6ï¼…and 86.93ï¼…, respectively. The predicted BrICEprotein includes a potential basic helix- loop-helix worked in concert to impartcold-regulated CBF expression, and several other domains. The secondary structureanalysis revealed that BrICE strongly resembled ICE1 and CbICE53. All results impliedthat BrICE might have the similar functions possessed by other ICE genes such asincluding the expression of some cold-regulated genes.4. Cloning and characterization of cold-related regulator BrLOS2.A regulator of other cold responsive gene BrLOS2 (Genbank accession numberDQ334724) was cloned from the roots of Brassica campestris ssp. chinensis, which werecold-induced for 12 hours. The full-length cDNA of BrLOS2 was 1666bp and contained a1335bp opening-reading frame(ORF) encoding 137.371kD peptide containing 444 aminoacids with pI of 4.97. Further comparison to Brassica rapa,Brassica napus,Arabidopsisthaliana,Capsella bursa-pastoris,Hevea brasiliensis and Spinacia oleracea LOS genesshowed the similarity were 94ï¼…, 96ï¼…, 91ï¼…, 90ï¼…, 80ï¼…and 83ï¼…, respectively. Thepredicted BrLOS2 protein contained enolase-N domain, enolase domain, conservedputative DNA-binding like LOS2 from Arabidopsis thaliana. The secondary structure andmolecular modeling analysis revealed that BrLOS2 strongly resembled other LOS genes.Cold acclimation assay revealed that BrLOS2 was expressed constitutively in Brassicacampestris ssp. chinensis and was involved in the cold acclimation process.5. The construction of plant expression vector Brassica campestris ssp.chinensis BrCOR14 geneWith gene specific primers designed from the full-length BrCOR14 gene, theopening-reading frame (ORF) was cloned from the leaves of Brassica campestris ssp. chinensis, which was cold-induced for 7 days. The amplified fragments were cloned topMD18-T easy vector, and it was confirmed by sequencing. The amplified fragments werecut by Bglâ…¡and BstEâ…¡enzymes, then inserted into expression vector pCAMBIA1304,giving an expressing plasmid containing the BrCOR14 gene. The recombinant vector wasintroduced into Agrobacterium tumefaciens with freeze-thaw method. The research hasgiven a basic theory and practice to future studies on the identifying the function ofBrassica campestris ssp. chinensis BrCOR14 gene.Through above Molecular Cloning and Characterization of four novel cold-responsivegenes and correlative regulators from Brassica campestris ssp. chinensis, we validated thatthere were cold-responsive genes BrCOR14,BrCBF,BrICE and BrLOS2 in Brassicacampestris ssp. chinensis. And the expressing of them was induced by cold. The BrLOS2expressed constitutively and up-regulated with time of cold induced.