毛竹PheMADS47a基因启动子克隆及功能分析

Cloning and Functional Analysis of the Promoter of PheMADS47a Gene in Moso Bamboo(Phyllostachys edulis)

MADS-box转录因子家族成员SVP作为开花调控网络的中枢调控因子,也可在植物芽发育中发挥作用并参与逆境及激素途径。为探究毛竹(Phyllostachys edulis)PheMADS47a基因启动子的功能,本研究克隆了PheMADS47a约2000 bp的启动子序列,构建了不同长度5′端缺失启动子(P1:1949 bp、P2:825 bp、P3:578 bp、P4:493 bp、P5:230 bp、P6:79 bp)的植物表达载体(含报告基因GUS),遗传转化至拟南芥(Arabidopsis thaliana),通过β-葡萄糖苷酸酶(GUS)染色分析其活性。结果显示,PheMADS47a基因启动子中存在脱落酸(ABA)、赤霉素(GA)、吲哚-3-乙酸(IAA)、茉莉酸甲酯(MeJA)、水杨酸(SA)等激素应答元件及低温、干旱等非生物胁迫响应元件以及众多光响应元件。在10和15 d幼苗期,除P4启动子外,其他长度的启动子片段均有活性;在23和33 d苗期,P1~P3启动子活性强。全长启动子P1驱动的GUS基因在拟南芥的根、茎、叶、花序、角果基部均有表达,而在角果和侧枝中无表达。PheMADS47a基因启动子活性明显受MeJA、SA促进,不同长度的启动子活性对ABA、GA和IAA的应答不同。不同长度的启动子活性均可被黑暗、干旱、NaCl、4℃低温抑制。推测-578~-493 bp是该启动子活性的关键区域,该启动子是MeJA诱导型启动子。本研究为应用PheMADS47a基因启动子和探究该基因的调控机制提供了理论依据。

SVP,a member of the MADS-box transcription factor,acts as a central regulator in plant flowering control network.It is involved in bud development and plays a role in abiotic stresses and hormone pathways.In this study,a 2000 bp segment of PheMADS47a gene promoter was cloned from Phyllostachys edulis.The full-length promoter and five 5′terminal truncations(P1:1949 bp,P2:825 bp,P3:578 bp,P4:493 bp,P5:230 bp,P6:79 bp)were fused with the GUS reporter gene to assess their activities by GUS staining in transgenic Arabidopsis thaliana.The results revealed the presence of hormone response elements such as ABA,GA,IAA,MeJA,and SA,as well as cis-acting elements linked to abiotic stresses such as low temperature and drought,and many light response elements within the promoter of PheMADS47a gene.All promoters except for P4 exhibited activity at both 10-day and 15-day seedling stages,while P1-P3 displayed activity at 23-day and 33-day seedling stages.GUS gene driven by full-length promoter P1 was expressed in roots,stems,leaves,flowers and bases of siliques but not in siliques or lateral branches in transgenic Arabidopsis thaliana.The promoter activity of PheMADS47a gene was notably promoted by MeJA and SA.Promoters with different lengths showed varying responses to ABA,GA and IAA,and could be inhibited by darkness,drought,NaCl and low temperature(4℃).It is speculated that-578 to-493 bp region is key to the promoter activity.The promoter is a MeJA-inducible.This study provides a theoretical basis for the utilization of PheMADS47a gene promoter and further study on the regulation of PheMADS47a gene.