水稻PHD17基因启动子克隆及冷胁迫应答分析

Promoter cloning and analysis of the cold-stress response of PHD17 gene in rice

  • 摘要: 目的 克隆水稻PHD(Plant homeodomain)蛋白基因OsPHD17的启动子序列,并进行冷胁迫应答分析,为深入探究OsPHD17基因的冷胁迫响应机制提供理论参考。方法 通过实时荧光定量PCR检测OsPHD17基因在冷胁迫下的表达模式,利用PlantRegMap数据库对其启动子转录因子识别元件进行预测,并克隆OsPHD17基因启动子序列(转录起始点上游1160 bp),连接至pCAMBIA3301载体上,从而获得植物表达载体(POsPHD17-GUS),通过根癌农杆菌介导转化拟南芥,获得转基因拟南芥株系。最后通过RT-PCR和实时荧光定量PCR检测在冷胁迫下的报告基因GUS表达水平,分析启动子在冷胁迫下的转录活性变化。结果 OsPHD17基因启动子包含参与调控生长发育的转录因子TALE、LBD、MIKC_MADS、G2-like和BBR-BPC等识别元件,以及冷胁迫应答相关的转录因子ERF、C2H2、bHLH、bZIP、NAC等多个识别元件。冷胁迫处理3.0 h时,OsPHD17基因相对表达量显著升高(P<0.05,下同),处理12.0 h时达到峰值,约为处理0 h的11.7倍,表明OsPHD17基因受到冷胁迫时可迅速作出应答。克隆获得的OsPHD17基因启动子序列长度为1160 bp,成功构建POsPHD17-GUS转基因拟南芥株系。通过RT-PCR检测发现,T3代纯合POsPHD17-GUS转基因拟南芥株系中GUS基因表达,说明OsPHD17基因启动子具有转录活性。对2株T3代纯合POsPHD17-GUS转基因拟南芥株系进行4 ℃胁迫处理,结果发现,与对照(未处理)相比,冷胁迫下2个株系GUS基因相对表达量极显著(P<0.01)或显著升高,表明OsPHD17基因启动子的转录活性升高。结论 克隆的OsPHD17基因启动子具有转录活性,可驱动下游基因转录,且在冷胁迫诱导下OsPHD17基因启动子转录活性升高,OsPHD17基因表达显著上调,证实OsPHD17基因在水稻冷胁迫应答中发挥重要的调控作用。

     

    Abstract: Objective This study aimed to clone promoter sequence of gene OsPHD17 in PHD (plant homeodomain) of rice and analyze its response to cold stress, so as to provide a theoretical reference for elucidating the cold-stress response mechanism of gene OsPHD17.Method Quantitative real-time PCR (qPCR) was employed to analyze expression pattern of gene OsPHD17 under cold stress treatment. Recognition elements of transcriptional factors of the promoter were predicted based on PlantRegMap database. The promoter sequence (length of 1160 bp upstream of the transcription start site) of gene OsPHD17 was cloned and inserted to vector pCAMBIA3301 to obtain plant expression vector POsPHD17-GUS. Transgenic Arabidopsis thaliana lines were generated via Agrobacterium tumefaciens-mediated transformation. Expression of GUS reporter genes under cold stress were detected by RT-PCR and qPCR to analyze the change in promoter transcriptional activity under cold stress conditions.Result The promoter of gene OsPHD17 included recognition elements of the transcriptional factors regulating growth and development (TALE, LBD, MIKC_MADS, G2-like, BBR-BPC) and recognition elements of the transcriptional factors regulating cold-stress response (ERF, C2H2, bHLH, bZIP, and NAC). Expression of the gene OsPHD17 was significantly up-regulated at 3.0 hours of cold stress (P<0.05,the same below); the expression peaked at 12.0 h with approximately 11.7-fold increase compared to the expression at 0 h, indicating a rapid cold-stress response of the gene OsPHD17. The cloned promoter sequence was 1160 bp in length, and transgenic Arabidopsis thaliana lines were successfully established. RT-PCR detection showed that T3 generation were homozygous with GUS genes expression of POsPHD17-GUS transgenic Arabidopsis thaliana lines, indicating that the promoter of gene OsPHD17 had transcriptional activity. Under cold stress of 4 ℃, the two homozygous T3 lines of Arabidopsis thaliana showed a highly significant (P<0.01) or significantly up-regulated relative expression of the GUS gene in the two lines compared to the CK group (untreated), demonstrating an increase of transcriptional activity of promoter of gene OsPHD17.Conclusion The promoter of gene OsPHD17 that exhibits transcriptional activity is capable of modulating downstream gene expression. Transcriptional activity is markedly upregulated under cold stress conditions, and expression of gene OsPHD17 is significantly upregulated, thereby confirming the important regulatory role of gene OsPHD17 in cold-stress response of rice.

     

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