小麦TaGAPC1基因及其启动子的克隆与功能分析  

Cloning and Function Analysis of TaGAPC1 Gene and the Promoter in Wheat(Triticum aestivum)

作　　者：秦凌月 邓西平[2] 杨淑慎[1] QIN Ling-Yue;DENG Xi-Ping;YANG Shu-Shen(College of Life Science,Northwest A＆F University,Yangling,712100,China;State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau/Institute of Soil and Water Conservation,Chinese Academy of Sciences,Yangling 712100,China)

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100 [2]中国科学院水土保持研究所/黄土高原土壤侵蚀和旱地农业重点实验室,杨凌712100

出　　处：《农业生物技术学报》2018年第10期1639-1649,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31671609);黄土高原土壤侵蚀与旱地农业国家重点实验室专项(No.10502)

摘　　要：甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)是糖酵解过程中的关键酶。作为GAPDH的亚型,GAPC(cytosolic glyceraldehyde-3-phosphate dehydrogenase)催化3-磷酸甘油醛氧化生成1,3-二磷酸甘油酸,但是关于GAPC在非生物胁迫应答中作用的研究却并不充分。本研究从中国春小麦(Triticum aestivum)中克隆出了TaGAPC1基因(GenBank No. KU246046),其编码337个氨基酸,并克隆出基因上游973 bp的序列,将其命名为P973。通过融合绿色荧光蛋白(green fluorescent protein,GFP)报告基因,使用基因枪法瞬时转化洋葱(Allium cepa)表皮细胞进行亚细胞定位,结果显示TaGAPC1蛋白定位于细胞膜上。使用实时荧光定量PCR(quantitative real-time PCR, qRT-PCR)技术,分析TaGAPC1基因在根、茎、叶中,以及干旱(PEG8000)、盐(NaCl)、脱落酸(abscisic acid, ABA)和低温(4℃)非生物胁迫下的表达模式。结果表明TaGAPC1基因在叶、根、茎中的表达量依次下降,在PEG8000、NaCl和ABA胁迫下的表达量显著上升,但对4℃的响应不明显。根据PLACE (http://www.dna.affrc.go.jp/PLACE/)和PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)数据库分析,P973启动子序列中包含响应干旱、ABA、低温和创伤等胁迫的顺式作用元件,如干旱应答元件(drought-responsive element,DRE)、激素应答元件包括ABA应答元件(ABA-responsive element, ABRE)、MYB结合位点(MYB-binding site, MBS)和WUN-motif等。根据顺式作用元件的分布,扩增得到5个启动子5'端缺失片段,分别命名为P844、P738、P605、P475和P256。构建6条启动子序列融合β-葡萄糖苷酸酶基因(β-glucuronidase, GUS)的表达载体,并瞬时转化烟草(Nicotiana batacum)植株,测定PEG8000、NaCl、ABA和4℃胁迫下各启动子驱动的GUS酶活。结果表明,-973^-605启动子区域在TaGAPC1基因应答PEG8000和NaCl胁迫中具有关键作用,-973^-475启动子区域对应答ABA胁迫至关重要。本研究从分子水平初�Glyceraldehyde-3-phosphate dehydrogenase（GAPDH） is a key enzyme in glycolysis, but the role of GAPC（cytosolic glyceraldehyde-3-phosphate dehydrogenase）, which is a cytosolic GAPDH isoform and catalyzes the conversion of 3-phosphoglyceraldehyde to 1,3-diphosphoglyceric acid, against abiotic stresses is largely unknown. In this study, the TaGAPC1 gene encoding 337 amino acids and the TaGAPC1 promoter named P973 were both cloned. We fused the TaGAPC1 to green fluorescent protein gene（GFP） and transformed it into onion（Allium cepa） epidermal cells, the result showed that TaGAPC1 protein localized on the cytomembrane. Quantitative real-time PCR was used to detect TaGAPC1 expression in leaf, root and stem or under PEG8000, NaCl, ABA, and 4 ℃ stresses, the results indicated that the expression levels of TaGAPC1 gene in leaf, root, and stem gradually declined, and TaGAPC1 expression was induced by PEG8000, NaCl, and ABA treatments, but it did not response to 4 ℃ treatment. The PLACE（http：//www.dna.affrc.go.jp/PLACE/）and PlantCARE（http：//bioinformatics.psb.ugent.be/webtools/plantcare/html/） database indicated that some cisacting elements responsive to abiotic stress were present in P973 promoter, such as Drought-responsive element（DRE）, ABA-responsive element（ABRE）, MYB-binding site（MBS） and WUN-motif. Based on the position of cis-acting elements in TaGAPC1 promoter, 5 deletions named P844, P738, P605, P475, and P256 were respectively amplified. After the 6 promoters were fused to β-glucuronidase gene（GUS） and transformation into tabacco（Nicotiana batacum） was performed, GUS activity driven by the 6 promoters under PEG8000, NaCl, ABA, and 4 ℃ stresses were determined. The result suggested that TaGAPC1 promoter region from-973 to-605 was essential for the response to PEG8000 and NaCl, the region from-973 to-475 was essential for the response to ABA. The study illustrated the relationship of TaGAPC1 gene with abiotic stress response at molecular level, and lay foundatio

关 键 词：甘油醛-3-磷酸脱氢酶(GAPDH) 非生物胁迫 启动子 顺式作用元件 

分 类 号：S512.1[农业科学—作物学]