Cloning and characterization of cold, salt and drought inducible C-repeat binding factor gene from a highly cold adapted ecotype of Lepidium latifolium L

Abstract

The dehydration-responsive element-binding (DREB) protein/C-Repeat Binding Factors (CBFs) belongs to APETALA2 (AP2) family transcription factors that binds to DRE/CRT cis-element in cold-responsive (COR) genes and induce COR genes. CBFs have been isolated and characterized from evolutionarily diverse plant species. CBF pathway is conserved by CBF regulon and the size or the number and kind of target genes vary among freezing sensitive and tolerant plants. Hence, cloning of CBFs from highly freezing tolerant plants such as Lepidium latifolium L. will be useful in understanding the freezing tolerance of this species. In this study, LlCBF, a CBF1 family gene from L. Latifolium L., was cloned using RT-PCR and RACE-PCR. The full length mRNA of LlCBF is 948 bp with an open reading frame of 642 bp, encoding a protein of 213 amino acids with a molecular weight of 23.92 kDa and a theoretical isoelectric point of 4.80. Amino acid sequence analysis showed that LlCBF has an AP2 DNA binding domain, a potential CBF type nuclear localization signal (NLS) and C-terminal acidic domain. Semi-quantitative RT-PCR analysis of LlCBF revealed that this gene is up-regulated by high salt, dehydration and low temperature stresses. The investigation is therefore successful in cloning of a gene having strong homology with CBF transcription factors and responsive to low temperature, high salt and dehydration conditions.

Keywords: Cold; Lepidium latifolium L.; LlCBF transcription factor; Salt and drought inducible.

Fig. 1

Nucleotide sequence and deduced amino-acid sequence of LlCBF. The putative nuclear localization signal (NLS) is singly underlined and AP2 domain is doubly underlined. Conserves motiffs are indicated in bold letters

Fig. 2

Multiple amino acid sequence alignment among the LlCBF protein with the corresponding protein sequences of other plants: Eutrema salsugineum DREB1 protein (GenBank accession number AAS00621), Thlaspi arvense cold TF (GenBank accession number ABV82985), Arabidopsis thaliana DREB1C (GenBank accession number AB007789), Chorispora bungeana CBF (GenBank accession number AAY21899), Arabis pumila CBF (GenBank accession number ABA42927), AtDREB1B (GenBank accession numberAB007788), AtDREB1A (GenBank accession number AB007787), Capsella bursa-pastoris DREB1A (GenBank accession number ABM21468), Nicotiana tabacum DREB1A (GenBank accession number ABD65969), Brassica juncea DREB1B (GenBank accession number ABX00639). “*” indicates positions which have a single, fully conserved residue; “:” indicates highly conserved positions, and “.” indicates the weaker conserved positions. Dashes indicate gaps in the amino acid sequences in order to optimize alignment. A thin line and thick line above amino acid sequence show the location of the YRG and RAYD elements in the AP2 domain, respectively

Fig. 3

Comparison of the conserved AP2 DNA binding domain sequences and structures of LlCBF and AtERF1(pdb ID 1gcca). In the aligned sequences, “*” indicates conservative amino acid residues. The three β-sheets and a α-helix were marked above the corresponding sequences. The amino acid residues that contact DNA were indicated in bold

Fig. 4

Phylogenetic tree of LlCBF and other CBF-like proteins: Nicotiana tabacum DREB1A (A.N. ABD65969), AtDREB1C (A.N. AB007789), Chorispora bungeana CBF (cbCBF) (A.N.AAY21899), AtDREB1B (A.N.AB007788), AtDREB1A (A.N. AB007787), Arabis pumila CBF (ApCBF) (A.N.ABA42927), Eutrema salsugineum DREB1 (EsDREB1) (A.N. AAS00621), Thlaspi arvense cold TF (Tacold) (A.N. ABV82985), Brassica juncea DREB1B (BjDREB1B) (A.N. ABX00639), Raphanus sativus CBF1 (RsCBF1) (A.N. ACX48435), Capsicum annum DREB (CaDREBLP1) (A.N. AY496155), Barley CBF3 (A.N. AF239616), OsDREB1A (A.N. AF300970), OsDREB1B (A.N.AF300972), Triticum aestivum CBF1 (TaCBF1) (A.N.AF376136), Glycine max DREBa (GmDREBa) (A.N. AY542886), Triticim aestivum DREB1 (TaDREB1) (A.N.AF303376), Atriplex hortensis DREB1 (AhDREB1) (A.N.AF274033), Medicago sativa DREB1 (MsDREB1) (A.N.ABY78835), Capsella bursa-pastoris DREB1A (CbDREB1A) (A.N. ABM21468)

Fig. 5

RT-PCR analysis of expression pattern of LlCBF transcript after treatment of cold stress (A), salt stress (B) and drought stress (C). Lepidium 26SrRNA gene act as internal control (D) to ensure equal amount of templates were added to each RT-PCR reaction. 0 h time point serves as control