银杏萜内酯合成关键酶基因启动子的功能解析

3-hydroxy-3-methylglutaryl coenzyme A reductase (GbHMGR) and 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (GbDXR) are two rate-limiting enzyme synthetic route for Ginkgo Terpene Lactones. Based on the two key gene promoter -- GbHMGR and GbDXR of the synthetic route for the cloned Ginkgo Terpene Lactones, the research analyzes its related functional element and possibly combinative transcription factor by using the Yeast one-hybrid technique combined with Chromatin Immunoprecipitation assay and Protein-DNA Binding Assay. By using Bisulfite Sequencing PCR and 5-aza treating analysis, it would be revealed that the effect of promoter methylation level on key gene expression regulation. Furthermore, the cis-acting element of the promoter of these two genes will be built into the plant expression vector, pBI121, that is lack of the 35s promoter. By using Agrobacterium-mediated method, it will be shifted to the tobacco. On the basis of the Gus expression and activity of the transgenic plants, in order to understand fully the mechanization that the cis-acting elements regulate the gene expression of GbHMGR and GbDXR, it analyzes the effect about the cis-acting element in the regulation of gene expression in time and space, and the specificity tissues from the induced expression for environmental factors and plant growth regulator. The research aims to reveal various cultivation measures regulate and control the gene expression of the Ginkgo GbHMGR and GbDXR how and then regulate accumulated molecule mechanism of the Ginkgo Terpene Lactones and plays the foundation for the molecular design breeding of the key gene of synthetic routine for Ginkgo biloba terpene lactones.

HMGR和DXR是银杏萜内酯合成途径中的两个关键酶。本研究在已克隆银杏萜内酯合成关键基因GbHMGR、GbDXR启动子的基础上，采用酵母单杂交技术、染色质免疫共沉淀及蛋白核酸互作分析其包含的功能元件及可能结合的转录因子与互作蛋白；启动子序列的BSP分析揭示甲基化对关键基因的转录调控影响；结合前期测序工作，采用数字表达谱分析转录因子与关键基因的调控网络；进一步将两个基因启动子包含的顺式作用元件构建到无35S 启动子的植物表达载体pBI121，通过农杆菌介导法转入烟草中，基于转基因植株的GUS 表达和活性检测，分析这些顺式作用元件受生长调节剂和环境因子诱导表达的特异性及调控基因时空表达机制。研究旨在揭示各种栽培措施调节银杏GbHMGR 和GbDXR基因表达进而调控银杏萜内酯积累的分子机理，为银杏叶萜内酯合成途径中关键基因的分子遗传育种奠定基础。

银杏萜内酯被认为是银杏叶片中关键的药用活性成分，银杏叶提取物中萜内酯成分和含量成为衡量产品质量的重要指标之一。阐明萜内酯代谢关键基因启动子顺式作用元件与相关转录因子在调控基因时空特异性表达及其响应不同环境因子诱导的分子机制，有助于探究不同调控措施调节银杏叶萜内酯含量的分子机理。. 本研究按照原计划完成了银杏GbHMGR、GbDXR启动子调控序列的分离及各顺式作用元件的功能分析。在转录组测序基础上，通过启动子序列与转录因子作用分析，得到了40多个GbWRKY转录因子和78个AP2/ERF转录因子家族，并进一步阐明了部分GbWRKY、AP2/ERF等转录因子响应环境因子及栽培措施，调控GbHMGR、GbDXR、GbMECP和GbMECT表达的分子机理，进一步阐述银杏萜内酯合成受环境因子及生长发育调控的分子机理，完成了原计划的研究工作。由于进一步研究的需要，本项目资助还额外完成了银杏GbMECP和GbMECT等启动子序列的分离及功能鉴定，为后期深入研究分析转录因子调控萜内酯合成的MVA和MEP两个不同路径分子机理奠定了基础。. 研究结果初步揭示了各种栽培措施调节银杏GbHMGR、GbDXR、GbMECP和GbMECT基因表达进而调控银杏萜内酯积累的分子机理，为银杏叶萜内酯合成途径中关键基因的分子遗传育种奠定基础。