Cloning Of A DREB Transcription Factor Gene From Sorghum

Abiotic stresses such as drought,salt and freezing could produce severe damages on growth and development of plants. Genes upregulated upon these stress conditions were basically classified into two groups:those that directly protect against environmental stresses,and those that regulate gene expression and signal transduction upon stress conditions. The first group of gene products includes transcription factors,protein kinases,and enzymes involved in phosphoinositide metabolism,such as bZIP, MYC,MYB,DREB,MAP, Phosphoric acid vinegar enzyme, et al.The second group includes proteins and enzymes that likely function by protecting cells from dehydration,such as late- embryogenesis-abundant (LEA)proteins,enzymes required for biosynthesis of proline,and aquaporins (AQP), et al, making the cell various physiological and biochemical activity can maintain normal metabolism. In the past few years,scientists have showed great interest in the second group of genes.In plants,the expression of many genes were regulated at transcriptional level,and mostly it's realized through cis- and trans-acting elements. Therefore,transcription factors could play a key role in stress prevention in plants.In this study, we obtained a DREB gene from Sorghum bicolor(L.) by RT-PCR using degenerate primers and RACE techonolgy. Through the multiply alignment of amino acid sequences and nucleotide acid sequences of several plant AP2/EREBP in GenBank, a pair of degenerate primers were designed against the conserved regions that was used for RT-PCR to amplify a 117 bp cDNA fragment from Sorghum bicolor(L.). Base on the sequence of the cDNA fragment, primers were designed for 3'RACE and a 949 bp cDNA fragment was amplified. It was named SbDREB1001, and was analyzed with relative bioinformatics methods. The DREB cDNA sequence was 949 bp long with an ORF (open reading frame) of 789 bp, encoding a predicted polypeptide of 262 amino acid with a calculated MW of 28.6 kDa and pI of 5.52. It contain a C-terminal hydrophobic domain. In addition, one AP2 domain between 83 and 138 amino acid. The amino acid sequence compared by NCBI_Blast revealed high homology with that of other plant DREB genes, and the similarity to ethylene response factor 2 of Zea mays(GeneBank Accession No.:NM-001158997.1)was 88%. The real-time fluorescence quantitative PCR was performed to reveal transcript level of DREB under different abiotic stresses. Real-time quantitative polymerase chain reaction (PCR) expression analysis results showed that DREB gene was involved in sorghum response to salt stress, but the response in speed was different in different materials under stress condiction.The response was 2 hours faster in 623B than HN16 under salt stress. The DREB gene insorghum play an important function under salt stress condition.