非生物胁迫下W-box突变对小麦TaGAPC5基因启动子活性的影响

摘要

小麦(Triticum aestivum)胞质甘油醛3-磷酸脱氢酶(cytosolic glyceraldehyde-3-phosphatedehydrogenase,GAPC) TaGAPC5基因在干旱等非生物胁迫下显著表达.序列分析表明,TaGAPC5基因启动子区包含3个W-box顺式作用元件.本实验利用PCR定点突变技术对TaGAPC5基因启动子区-640处W-box单独突变、-640、-700两处W-box突变和-640、-700、-997处W-box均突变,将这3种突变的启动子分别与植物表达载体pC0390-GUS融合表达,利用农杆菌(Agrobaoterium tumefaciens)介导法转化烟草(Nicotiana tabacum),对转化后的烟草分别进行正常条件下培养和100 nmol/L脱落酸(abscisic acid,ABA)、20％ PEG8000胁迫处理.组织化学染色结果显示,这3种突变后的启动子序列均具有一定的启动子活性.烟草叶片GUS酶活检测结果表明,在正常生长条件下,TaGAPC5基因启动子-640处W-box单独突变对启动子活性影响不大,-640、-700、-997三处W-box均突变TaGAPC5基因启动子活性显著降低(P＜0.01);在1 00 nmol/L ABA诱导下,-640处W-box单独突变、-640、-700两处W-box突变和-640、-700、-997处W-box均突变均可显著降低TaGAPC5基因启动子活性(P＜0.01);在PEG胁迫条件下,TaGAPC5基因启动子-640处W-box单独突变和-640、-700、-997三处W-box均突变可显著降低该基因启动子活性(P＜0.01).因此表明-640、-700、-997处W-box对小麦TaGAPC5基因启动子活性有一定影响,其中在1 00 nmol/L ABA、20％ PEG8000胁迫条件下其影响较大,推测TaGAPC5基因启动子序列中W-box元件可能参与非生物胁迫下小麦TaGAPC5基因的表达调控.这一研究结果可为小麦TaGAPC5基因在非生物胁迫下的表达调控研究提供重要线索.%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.