Abstract：In wheat (Triticum aestivum), the cytosolic glyceraldehyde-3-phosphate dehydrogenase gene TaGAPC5 was induced under abiotic stresses. Sequence analysis showed that the TaGAPC5 gene promoter region contained three W-box cis-elements. In this study, PCR-mediated mutagenesis method was performed to introduce -640 site mutagenesis, -640 and -700 both sites mutagenesis, and -640 and -700 and -997 three sites mutagenesis in TaGAPC5 gene promoter region. Then, recombinants of the three mutagenesis sequences with pC0390-GUS vector were constructed, respectively, and transformed into Nicotiana tabacum by Agrobacterium-mediated approach. The transformed tobaccos growth under normal conditions and were treated with 100 nmol/L abscisic acid (ABA) and 20% PEG8000, respectively. Histochemical staining results showed that the three mutations of TaGAPC5 promoter all had promoter activity. The results of GUS enzyme activity in tobacco leaves revealed that under normal growth condition, compared with the TaGAPC5 gene promoter without mutagenesis，-640 alone site mutant didn't affect the activity of the promoter, the activity of mutant at -640 and -700 and -997 three sites was decreased significantly(P＜0.01). What's more, under the 100 nmol/L ABA treatment, -640 site mutant, -640 and -700 both sites mutant, and -640 and -700 and -997 three sites mutant all significantly reduced the activity of the TaGAPC5 gene promoter (P＜0.01); under 20% PEG8000 treatment, the activities of -640 alone site mutant and the -640, -700, -997 mutant were both decreased more significantly (P＜0.01). Taken together, the results proved that -640, -700, -997 three W-box sites in TaGAPC5 gene promoter region had a great role in promoter activity, particularlly in 100 nmol/L ABA、20% PEG8000 conditions, and suggested that the W-box cis-elements of TaGAPC5 gene promoter may participate in the regulation of TaGAPC5 gene expression under abiotic stress. Thus, the results of this study could provide important clues for regulatory mechanism of TaGAPC5 gene under abiotic stress in wheat.
于丽丽, 高彩球, 王玉成,等. 2010. 桂柳甘油醛-3-磷酸脱氢酶基因的克隆与表达分析. 东北林业大学学报, 38(07):105-108. (Li L Y, Cai Q G, Yu C W, et al. 2010. Cloning and expression of a glyceraldehyde-3-phosphate dehydrogenase gene from Tamarix hispida[J].Journal of northeast forestry university, 38(07):105-108.)齐晓花, 许学文, 罗晶晶, 等. 2011. 黄瓜 3-磷酸甘油醛脱氢酶基因 CsGAPDH 的克隆及其涝胁迫响应分析. 园艺学报, 38(9):1693-1698.(Xiao H Q, Xue W X,Jing J L,et al. 2011. Cloning,characterization and expression of CsGAPDH,a waterlogging responsive gene in cucumber[J]. Acta Horticulturae Sinica, 38(9):1693-1698.)田畅, 蒋倩, 王枫, 等. 2014. 3个芹菜品种 Agnp-G3PDH 基因的克隆及其序列和表达特性分析. 植物资源与环境学报, 23(2):1-10.(Chang T, Qian J, Feng W, et al. 2014. Cloning of Agnp-G3PDH gene from three cultivars of Apium graveolens and its sequence and expression characteristic analyses[J]. Journal of Plant Resources and Environment,23(2):1-10)李晓泽, 刘关君, 杨传平. 2007. 西伯利亚蓼甘油醛-3-磷酸脱氢酶基因的 cDNA 克隆与序列分析. 植物生理学通讯, 43(1):41-48.(Xiao Z L, Guan J L , Chuan P Y. 2007.Cloning and sequence analysis of cDNA of glyceraldehyde-3-phosphate dehydrogenase gene from Polygonum sibiricum Laxm[J].Plant Physiology Communications, 43(1):41-48.)张梅娟, 沙伟, 刘博, 等. 2014. 东亚砂藓甘油醛3-磷酸脱氢酶基因的克隆及表达分析. 安徽农业科学, 42(25):8506-8510.(Mei J Z,Wei S, Bo L,et al. 2014. Cloning and expression analysis of glyceraldehyde-3-phosphate dehydrogenase gene RjGAPDH in Racomitrium japonicum[J]. Journal of Anhui Agriculture Science, 42(25):8506-8510.)张旸, 郭海俊, 刘龙彪, 等. 2014. 星星草 PtGAPDH 基因的克隆与表达分析. 草业学报, 23(2):207-214.(Yang Z, Hai J G, Long B L, et al. 2014. Cloning and expression analysis of PtGAPDH in Puccinellia tenuiflora[J]. Acta Prataculturae Sinica, 23(2):207-214)张琳, 邓荣, 马兰兰, 等. 2014. 小麦3-磷酸甘油醛脱氢酶基因(GAPDH)上游序列的克隆及功能验证. 农业生物技术学报, 22(8): 973-982.(Lin Z, Rong D, Ma L L,et al. 2014.Cloning and function verification of glyceraldehyde-3-phosphate dehydrogenase gene(GAPDH) upstream sequence from Wheat(Triticum aestivum L.)[J]. Journal of Agricultural Biotechnology,22(8): 973-982.).Bae M S, Cho E J, Choi E Y, Park O K. 2003. Analysis of the Arabidopsis nuclear proteome and its response to cold stress[J]. Plant Journal, 36: 652-663.Baek D, Jin Y, Jeong J C, et al. 2008. Suppression of reactive oxygen species by glyceraldehydes-3-phosphate dehydrogenase[J]. Phytochemistry, 69:333-338.Eulgem T, Somssich IE 2007. Networks of WRKY transcription factors in defense signaling[J]. Current Opinion in Plant Biology 10(4): 366-371.Guo L, Devaiah S P, Narasimhan R, et al. 2012. Cytosolic glyceraldehydes-3-phosphate dehydrogenases interact with phospholipase D delta to transduce hydrogen peroxide signals in the Arabidopsis response to stress[J]. Plant Cell, 24(5): 2200-2212.He G, Xu J, Wang Y, et al. 2016. Drought responsive WRKY transcription factor genes TaWRKY1 and TaWRKY33 from wheat confer drought and/or heat resistance in Arabidopsis[J]. BMC Plant Biology, 16:116.Jefferson R A, Kavanagh T A, Bevan M W. 1987. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[J]. The EMBO Journal. 6(13):3901-3907.Jeong M J, Park S C, Byun M O. 2001. Improvement of salt tolerance in transgenic potato plants by glyceraldehydes-3-phosphate dehydrogenase gene transfer[J]. Molecular Cell, 12(2): 185-189.Jiang Y, Yang B, Harris N S, et al. 2007. Comparative proteomic analysis of NaCl stress responsive proteins in Arabidopsis roots[J]. Journal of Experimental Botany, 58(13): 3591-3607.Kappachery S, Hiremath G B, Yu J W, et al. 2015. Effect of over and under expression of glyceraldehydes-3-phosphate dehydrogenase on tolerance of plants to water deficit stress[J]. Plant Cell Tissue and Organ Culture., 121:97-107.Laxalt A M, Cassia R O, Sanllorenti P M, et al.1996. Accumulation of cytosolic glyceraldehydes-3-phosphate dehydrogenase RNA under biological stress conditions and elicitor treatments in potato[J]. Plant Molecular Biology, 30:961-972.Li S J, Fu Q T, Chen L G,et al. 2011. Arabidopsis thaliana WRKY25, WRKY26, and WRKY33 coordinate induction of plant thermotolerance[J]. Planta, 233(6): 1237-1252Li SJ, Zhou X, Chen L G, et al. 2010. Functional characterization of Arabidopsis thaliana WRKY39 in heat stress[J]. Molecules and Cells, 29(5): 475-483Manjunath S, Sachs M M. 1997. Molecular characterization and promoter analysis of the maize cytosolic glyceraldehydes-3-phosphate dehydrogenase gene family and its expression during anoxia[J]. Plant Molecular Biology, 33(1):97-112.Nezhadahmad A, Prodhan Z H, Faruq G. 2013. Drought tolerance in wheat[J].Scientific World Journal, 3:1-12Niu C F, Wei W, Zhou Q Y, et al. 2012. Wheat WRKY genes TaWRKY2 and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis plants[J]. Plant Cell and Environment, 35:1156–70Okay S, Derelli E, Unver T. 2014. Transcriptome wide identification of bread wheat WRKY transcription factors in response to drought stress[J]. Molecular Genetics and Genomics,289(5): 765-781.Pelah D, Shoseyov O, Altman A, et al. 1997. Water stress response in Aspen (Populus tremula): differential accumulation of dehydrin, sucrose synthase, GAPDH homologues, and soluble sugars[J]. Journal of Plant Physiology, 151(1):96-100.Qin Y, Tian Y, Liu X. 2015. A wheat salinity-induced WRKY transcription factor TaWRKY93 confers multiple abiotic stress tolerance in Arabidopsis thaliana[J]. Biochemical and Biophysiology Research Communication, 464(2):428-433Roth U, von Roepenack Lahaye E, Clemens S. 2006. Proteome changes in Arabidopsis thaliana roots upon exposure to Cd2+[J]. Journal of Experimental Botany, 57(15):4003-4013.Russell D A, Sachs M M. 1989. Differential expression and sequence analysis of the maize glyceraldehydes-3-phosphate dehydrogenase gene family[J]. Plant Cell, 1(8): 79-803.Shang Y, Yan L, Liu Z Q, et al. 2010. The Mg-Chelatase H subunit of Arabidopsis antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition. Plant Cell, 22(6): 1909-1935.Sparkes I A, Runions J, Kearns A , et al. 2006. Rapid, tran-sient expression of fluorescent fusion proteins in tobac-co plants and generation of stably transformed plants[J].Nature Protocols, 1(4): 2019-2025.Ulker B, Somssich IE 2004 WRKY transcription factors: from DNA binding towards biological function[J]. Current Opinion in Plant Biology, 7(5):491-498.Velasco R, Salamini F, Bartels D. 1994. Dehydration and ABA increase mRNA levels and enzyme activity of cytosolic GAPDH in the resurrection plant Craterostigma plantagineum[J]. Plant Molecular Biology, 26(1):541-546.Wang C, Deng P, Chen L, et al. 2013. A Wheat WRKY transcription factor TaWRKY10 confers tolerance to multiple abiotic stresses in transgenic tobacco[J]. PLoS One 8(6): e65120. Wang X, Zeng J, Li Y, et al. 2015. Expression of TaWRKY44, a wheat WRKY gene, in transgenic tobacco confers multiple abiotic stress tolerances[J]. Frontiers in Plant Science,6:615Wu K L, Guo Z J, Wang H H, et al. 2005. The WRKY family of transcription factors in rice and Arabidopsis and their origins[J]. DNA Research,12:9-26.Yang Y, Kwon H B, Peng H P, et al. 1993. Stress responses and metabolic regulation of glyceraldehydes-3-phosphate dehydrogenase genes in Arabidopsis[J]. Plant Physiology, 101(1):209-16.Zeng L, Deng R, Guo Z, et al. 2016. Genome wide identification and characterization of glyceraldehyde-3-phosphate dehydrogenase genes family in wheat (Triticum aestivum) [J]. BMC Genomics, 17(1):240.Zhang X H, Rao X L, Shi H T, et al. 2011. Overexpression of a cytosolic glyceraldehydes-3-phosphate dehydrogenase gene OsGAPC3 confers salt tolerance in rice[J]. Plant Cell Tissue and Organ Culture, 107(1): 1-11.Zhang Z L, Xie Z, Zou X L,et al. 2004. A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells[J]. Plant Physiology, 134(4): 1500-1513.
