Gene Cloning of HaDREBA5 from Sunflower (Helianthus annuus) and Its Responses to Biotic and Abiotic Stress
SUN Rui-Fen1,*, ZHANG Yan-Fang2, NIE Li-Zhen1, NIU Su-Qing1, HAN Ping-An1, GENG Mu-Dan3, CHANG Yue1, TANG Kuan-Gang1
1 Inner Mongolia Academy of Agricultural ＆ Animal Husbandry Sciences, Huhhot 010031, China; 2 College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Huhhot 010011, China; 3 Inner Mongolia Horticulture Research Institute, Huhhot 010010, China
Abstract：Dehydration-responsive element binding protein (DREB) belongs to a subfamily of ethylene response factor (ERF) family. The cloning and functional analysis of DREB gene family is helpful for further revealing the regulation mechanism of APETALA2 (AP2)/ERF superfamily transcription factors in sunflower (Helianthus annuus) under biotic and abiotic stress. Based on the known sequence obtained from salt-induced differentially expressed genes in sunflower, the 3' and 5' ends of the sequence were amplificated and assembled into full cDNA sequence of 847 bp (GenBank No. MH085932). The cDNA contained an open reading frame of 468 bp which encoded 155 amino acids. The isoelectric point and molecular weight of the protein was predicted to be 5.21 and 37.5 kD, respectively. The amino acid sequence contained a conserved AP2/ERF DNA binding domain, including conserved YRG element and WLG motif, and the 14th and 19th amino acids in the AP2/ERF binding domain was valine and glutamate, respectively, implying that the gene belonged to DREB subfamily. Evolutionary analysis of transcription factors of AP2/ERF subfamily in Arabidopsis thaliana further indicated that the transcription factor belonged to A5 group of DREB subfamily．Blast analysis showed that the gene had a high similarity with many reported nucleotide sequences and deduced amino acid sequences of ERF family members in other plants. The full-length gDNA sequence of the gene was cloned by PCR method according to the full-length cDNA sequence of HaDREBA5 with the length of 468 bp from start codon to stop codon (GenBank No. MH085933), which had no introns. qRT-PCR study indicated that the expression of HaDREBA5 in sunflower could be induced by pathogen, mechanical damage, low temperature, NaCl and salicylic acid stress and showed different expression patterns under different types of stress. HaDREBA5 was expressed in roots, hypocotyls and leaves of sunflower and had organ expression specificity. The subcellular localization displayed that it was distributed in the nucleus of onion (Allium cepa) cells. The overexpression of HaDREBA5 in tobacco (Nicotiana tabacum) plants could enhance the tolerance to low temperature, drought and salt stress. Under NaCl stress, both the content of chlorophyll, soluble protein and proline and the activity of peroxidase (POD) and superoxide dismutase (SOD) increased; the expression of stress-related genes such as P5CS (Δ′-pyrroline-5-carboxylate synthetase), POD, MnSOD and GuZnSOD were also up-regulated in transgenic tobacco plants. The cloning and functional study of HaDREBA5 gene will help to understand the molecular mechanism of sunflower stress resistance, and provide gene resources and reference for stress resistance breeding in sunflower or other crops.
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