title: Identification and expression analysis of CBF/DREB1 and COR15 genes in mutants of Brassica oleracea var. botrytis with enhanced proline production and frost resistance.
abstract: Frost resistant mutants of Brassica oleracea var. botrytis were investigated for the presence of CBF/DREB1 and COR15a gene products and induced frost resistance. Total RNA of clones was isolated after 3 h, 6 h, 24 h and 14 d acclimation at 4 °C and proteins and free proline were isolated after 14 d acclimation. cDNA was produced using RT-PCR and the first CBF gene in B. oleracea detected and did quantify. Through SDS-PAGE and Western blotting, the COR15a protein was detected for the first time in B. oleracea. The results confirmed the first report of the presence of BoCBF/DREB1 in B. oleracea and this only appeared under cold acclimation. The sequence analysis of predicted amino acids revealed a very high homology (90%) with CBF sequences of other Brassica species (BnCBF5/DREB1, BrDREB1 and BjDREB1B) and homology reduced to 67% when compared to plants other than Brassicas. BoCBF/DREB1 transcript levels increased up to 24 h acclimation and then declined. Some mutants showed BoCBF/DREB1 expression at 3 h while others only after 6 h and 24 h acclimation. The genotypes showed positive significant correlation between BoCBF/DREB1 expression and frost resistance (R(2) = 0.9343). The proline level under acclimation increased about 8 fold and demonstrated positive and significant correlation with BoCBF/DREB1 expression. Proline also showed positive and significant correlation with frost resistance under cold acclimation but very not under non-acclimation. All clones were positive for COR15a protein after 14 d cold acclimation and expression correlated with frost resistance. Under non-acclimation COR15a was constitutively expressed in 3 mutants.
authors: Department of Biotechnology, University of Malakand, KPK, Pakistan. fazalbiotech@yahoo.com
authors: Fazal
authors: F
authors: Hadi
authors: Martyn
authors: M
authors: Gilpin
authors: Michael P
authors: MP
authors: Fuller
mesh_terms: D000064:Acclimatization / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001937:Brassica / Q000235:genetics / Q000502:physiology*; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D005615:Freezing; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D018515:Plant Leaves / Q000235:genetics / Q000502:physiology; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011392:Proline / Q000032:analysis / Q000378:metabolism*; D018749:RNA, Plant / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013223:Statistics as Topic; D013312:Stress, Physiological; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D018749:RNA, Plant; D014157:Transcription Factors; D011392:Proline