截形苜蓿(Medicago truncatula)Dof 32基因启动子克隆及功能区缺失分析

Promoter Cloning of Dof 32 Gene and Deletion Analysis of Its Functional Region in Alfalfa(Medicago truncatula)

Dof (DNA binding with one finger)基因家族是植物特有的一类转录因子,参与植物生长发育过程中种子的萌发、休眠及植物的开花等过程,在逆境响应中起到调控作用。为了研究截形苜蓿(Medicago truncatula) Dof 32启动子的组成及功能,以截形苜蓿DNA为模板,进行启动子片段扩增,预测启动子相关转录因子的结合位点。据此对5’端序列进行缺失克隆,构建相应的表达载体并转化拟南芥,通过GUS染色和实时荧光定量分析各功能区缺失启动子在自然条件下和盐胁迫条件下的生物学活性,探索启动子核心区域及顺式作用元件的功能。结果表明:除DPS3F启动子缺失片段活性偏弱外,全长片段DP32及其余2个缺失片段DPS1F、DPS2F的活性均较强,能够驱动下游GUS基因的表达。在盐胁迫条件下DP32、DPS1F、DPS2F转基因拟南芥GUS基因的表达量较自然条件下显著上升。本研究为探索Dof基因在截形苜蓿抗逆过程中的调控机理提供资料。

Dof(DNA binding with one finger) gene family is a special class of transcription factor that participates in the processes of seed germination, dormancy, and flowering of plants during plant growth and defense responses.To study the composition and function of promoter of Dof 32 gene, DNA of Medicago truncatula was used as the templates, predict the binding site of the promoter-associated transcription factor. Accordingly, the 5’-end sequence was missing cloned, and the corresponding expression vector was constructed and transformed into Arabidopsis thaliana, biological activity of each deletion vector under natural conditions and salt stress conditions by GUS staining and real-time fluorescence quantitative analysis, explore the core region of the promoter and the functions of cis-acting elements. The results showed that except for the weak activity of the DPS3 F promoter deletion fragment, the full-length fragment DP32 and the other two deletion fragments which means DPS1 F and DPS2 F were more active, which could drive the expression of the downstream GUS gene. GUS expression of DP32, DPS1 F and DPS2 F in transgenic Arabidopsis thaliana was significantly increased under salt stress than under natural conditions. This study provides basic data for the regulation of Dof gene regulation in the process of Medicago truncatula resistance to stress.