白及液体悬浮培养合成9,10-二氢菲的诱导调控和机制研究

Bletilla Striata, as a well-known and endangered perennial medicinal plant, is widely used in pharmacy and cosmetic industries. With the expanding uses of compounds of this medicinal plant, the traditional production mode can not meet the requirement, science the output from the planting in the open field is mostly depended on many uncontrolled conditions. Plant bioreactor system can greatly raise the productivity on plant cells and their secondary metabolites, which could yield great prospect of commercial application. During the former studies on cell suspension culture system construction for producing 9,10-dihydrophenanthrenes (DHP), we found that the medium ingredients and the factors of culture conditions resulted in significant changes in production of cells and the DHP. How the results are affected by these ingredients and factors, and what kinds of genes are involved in these processes have remained uncovered. In this study, the Response Surface Method is to be used for screening the optimum values for these ingredients and factors, which could help to construct the culture system with vigorous cell proliferation and highly expressed level of DHP. At the mean time, an optimized protocol is to be designed to establish for efficient separation of DHP from the culture system. Finally, a micro-fluid miRNA array which has been developed by our team will be applied for detecting the miRNAs and target mRNAs involved in cell proliferation and DHP expression. This work is firstly designed as a technique manual for bioactor construction of B. Striata and adjustment, and it takes relatively precise clues for key genes’ involved in regulation of cell proliferation and DHP expression. These expected results could pay an important theoretical and experimental foundation for application of B. Striata bioreactor and for production of DHP in suspension culture.

白及是重要的医药与工业原料，通过传统的田间种植来获取其药用活性成分已不能满足市场需求，运用植物生物反应器转变白及细胞及其次生代谢物的生产方式，能极大地提高生产效率，具有重要的商业前景。本项目组在构建白及细胞悬浮培养技术体系表达9,10-二氢菲（DHP）时，发现培养基配方和培养环境等因素对细胞增殖和DHP积累有显著影响，但这些因素的影响机制及基因调控过程尚未得知。本项目拟通过响应面优化设计，探索相关因素对白及细胞增殖和DHP合成的条件控制，构建高水平表达DHP的白及生物反应器培养体系，建立从该体系中高效分离DHP的方法，用已建立的微流体原位合成植物miRNA芯片鉴定出参与细胞增殖和DHP合成的miRNA及其靶基因变化规律，探索高水平表达DHP的机制。本研究结果可为构建白及生物反应器生产DHP提供技术支持，也可为白及细胞增殖和DHP合成关键调控基因的揭示提供实验基础。

以贵州道地的优势白及资源为研究材料，整合两个合作单位前期的相关研究基础和优势，本项目开展了如下几个方面的工作：（1）应用生物反应器高通量培养白及种苗，分析不同因素对种苗质量的影响，优化反应器和种苗培养的参数；（2）筛选和优化液体悬浮环境下白及愈伤的诱导、培养条件，构建起愈伤增殖的数学模型，进一步在该优化体系下，检测9,10-二氢菲等4种此生代谢产物生物合成的最佳体系和数学模型，描述其合成积累的动力学特征；（3）首次构建起涵盖白及全生育期不同组织基因组转录的核苷酸数据库，对该数据库进行生物信息学注解和EST-SSR分子标记的开发，并成功对新开发的分子标记进行了验证和应用。相关研究结果为阐释白及9,10-二氢菲化合物和特征性次生代谢物生物合成途径的分子机制以及白及细胞株系遗传改良等后续研究奠定了良好的研究基础。共申报授权专利9项，其中发明专利4项，实用新型专利5项；发表论文15篇，其中SCI收录6篇，北图核心收录8篇；培养硕士研究生6名，其中已毕业3名；申报科研项目多项，其中获资助2项；参加学术会议和做报告10人次；完成1人的职称晋升。总体而言，本项目超额完成了项目预期成果。