刚毛柽柳ThDof转录因子调控盐胁迫响应的分子机理研究

DNA-binding with one finger (Dof) domain proteins are a group of plant-specific transcription factors that are associated with many biological processes. In the previously study, we identified twenty Dof genes (designated as ThDof1-20) with complete open reading frame (ORF) from the salt stressed Tamarix hispida transcriptomes. Moreover, we had confirmed that four ThDof genes (ThDof 2, 3 ,6 and 14) played a role in the response to salt stress. On this basis, we will analyze the salt-tolerance mechanisms regulated by the four ThDofs on both physiological and molecular level in this project. .Firstly, the ThDofs promoter sequences will be cloned and pThDofs::GUS gene fusion construct is generated to investigate the expression patterns of ThDofs gene under salt stress. The cis-acting elements involved in regulation by salt stress are analyzed and selected for the further yeast one-hybrid assays to determine the upstream regulators of ThDof..Secondly, we will generate the transgenic Tamarix plants with ThDof-overexpression and silence and compare the physiological changes of salt tolerance conferred by ThDof. Furthermore, the differential expression genes between the ThDof-overexpression and silence transgenic Tamarix plants are identified by gene expression profiles. And we will further investigate metabolic pathway involved by different genes. Our study will contribute to salt tolerance regulation mechanism of woody plants.

Dof蛋白是植物所特有的一类转录因子，参与多个信号途径的调控。我们从盐胁迫柽柳转录组中分离获得20个全长ThDof基因，并通过研究证明其中的4个ThDof基因参与了柽柳的耐盐胁迫应答。在此基础上，本项目拟进一步系统研究柽柳ThDof基因耐盐生理功能及其分子调控机制。克隆ThDof基因的启动子序列并与GUS融合，阐明ThDof转录因子的耐盐时空表达模式；鉴定启动子中盐胁迫响应的顺式作用元件，通过酵母单杂交系统研究其上游表达调控通路；培育ThDof基因过表达和沉默柽柳，分析ThDof基因过表达和沉默对树木生理水平的调节。并进一步利用基因表达谱技术，鉴定ThDof基因所调控的下游靶基因，分析这些基因的产物所参与或调节的代谢过程，来阐明ThDof转录因子的分子生物学功能。预期结果对完善木本植物的耐盐调控机制具有重要意义。

Dof 蛋白是植物所特有的一类转录因子，参与多个信号途径的调控。本研究克隆获得1874 bp柽柳ThDof基因启动子序列，序列分析表明，该启动子区包含多种与逆境响应相关的顺式作用元件，如DOF、WBOX、ABRE等。将ThDof 基因启动子置换载体pCAMBIA1301上35S启动子驱动GUS基因。对转基因Pro ThDof::GUS拟南芥的GUS染色，结果显示ThDof基因在胚根和根中高表达，幼叶中的表达高于成熟叶，而在种子和荚果中表达量很低。不同胁迫处理柽柳ThDof基因启动子表达分析则显示，ThDof基因启动子能对NaCl、Mannitol胁迫和ABA处理做出应答。. 进一步将启动子分成了2段，分别置换载体pCAMBIA1301上35S启动子驱动GUS基因。GUS活性测定结果表明-1227~-1有启动子活性，而-580~-1无启动子活性，其他启动子片段的活性分析，还在研究中。.　对柽柳组培体系进行了优化，确定了NAA浓度为0.05 mg/L，6-BA为1 mg/L为最佳芽分化培养基。而继代和生根培养基使用1/2MS培养基。此外，建立了柽柳转基因体系，并获得了过量表达和抑制表达ThDof基因柽柳。瞬时转化柽柳结果表明过表达ThDof基因柽柳提高了GST和POD酶活性，MDA和相对电导率降低。本研究为进一步分析ThDof转录因子的抗逆功能及机制奠定了基础。