| 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步阐释了 TaGAPC1与
非 生物胁迫应答的关系，为深入探讨其抗逆的分子机制奠定了基础。 |
| 其他摘要 | 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 islargely 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 cis -
acting 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 foundation for further exploring TaGAPC1 's molecular mechanism
under stress. |
修改评论