蛇足石杉孢子萌发控制因子及生活史研究

Huperzia serrata (Thunb.ex Murray) Trevs. is the major material of huperzine A drugs for Alzheimer's disease, but the drugs are not produced on large scale because of the shortage of the wild resources. We found the spore propagation may be potential way to provide enough resources in the future and protect the nature population. But now, the technology of spore propagation in sterile medium is not set up. Therefore, three aspects will be the emphasis in this project, and they are as following: learning the structures of spore, exploring the key factors of spore germination and revealing the processes of gametophyte development and the lifecycle of Huperzia serrata. The material of Huperzia serrata will be collected in two populations, which are Huangshui town of Shizhu county in Chongqing province and Bawangling Nature reserve of Hainan Province in China. The habitat factors of these two populations will be obtained by ecology investigation, and the optimal factors will be determined for culture spore. The electron micrograph technology will be used to observe the structures of spore from natural populations and cultured spores treated by different light color, ultrasonic wave, gibberellins and enzyme. And the best way will be found to accelerate germination. The contents of gibberellins, abscisic acid and polyphcenols in spores and mediums of culturing spore by ELISA method and ultraviolet-visible absorption spectroscopy. The inhibit factors of spore germination may be filtered by detecting the concentration of acid radical in habitat soil with the method of Ion Chromatography with Spectrophotometric Detection. We will culture the spore in aseptic medium to observe the process of gametophyte development and complete the whole species lifecycle. This project will provide the foundation to reveal the origin of inhibitory substance and further study the mechanism of inhibition. These studies will be in favor of establishing the system of spore propagation on large scale.

蛇足石杉是治疗老年痴呆的石杉碱甲类药品的主要原材料，该物种资源短缺成为限制药品生产的瓶颈，孢子繁殖可能是解决资源短缺的潜在方法，但孢子萌发技术至今尚未突破。为此，我们以了解孢子结构、探寻孢子萌发控制因子和弄清配子体发育及生活史过程为重心，开展以下研究：利用生态学调查获得重庆黄水镇和海南霸王岭自然保护区两蛇足石杉居群的生境因子，确定孢子萌发的理想环境；利用显微成像技术观察孢子壁结构及光色、超声、赤霉素、生物酶等破壁技术的优劣；采用酶联免疫法及紫外分光光度法测定孢子及其培养液中赤霉素、脱落酸和多酚含量，并用离子色谱法筛选土壤中影响孢子萌发的酸性物质；同时开展孢子无菌培养，弄清配子体发育及物种生活史过程。本研究旨在掌握影响孢子萌发的关键环境因子、关键控制物质及最佳的破壁方法，绘制蛇足石杉完整生活史。本项目为今后探寻孢子萌发抑制物质来源及抑制机制奠定基础，并为建立孢子繁育技术体系提供技术支撑。

蛇足石杉 因其所含的石杉碱甲对治疗老年痴呆（AD）效果显著且毒副作用小而广受世人关注，但目前已野生资源已远不能满足市场需求。根据前期研究发现孢子繁殖有可能是实现规模化生产的潜在途径，但如何实现孢子萌发，培育出孢子幼苗是当前亟待解决的难题。本课题组在前期研究的基础上，确定了两个蛇足石杉的代表居群，即以孢子繁殖为主要种群更新方式的重庆石柱县黄水镇蛇足石杉居群和以芽胞繁殖为主要种群更新方式的海南昌江霸王岭居群。本项目主要针对这两居群开展了以下研究，试图找出影响孢子萌发的关键因子，掌握孢子萌发技术，弄清蛇足石杉生活史：1、收集两居群的空气温湿度；2、测定两地蛇足石杉中石杉碱甲含量差异；3、检测了两居群土壤中蛋白酶、多酚氧化酶、过氧化氢酶、磷酸酶、纤维素酶和蔗糖酶等7种酶活性；4、检测两居群中不同生长阶段蛇足石杉植株中赤霉素、生长素、水杨酸、脱落酸及细胞分裂素含量；5、采用TTC法检测孢子活力；6、采用不同处理方式培养孢子，并实时观察；7、野外采集孢子、配子体及孢子幼苗等进行形态观察,弄清蛇足石杉生活史；8、分离、培养配子体表面真菌，并进行鉴定；以带有上述真菌的培养基培养培养孢子，观察孢子萌发情况。结果表明蛇足石杉最适宜生长温度为20℃，适度90%以上；重庆居群的蛇足石杉中石杉碱甲含量远高于海南样地；重庆样地土壤蛋白酶活性、过氧化氢酶、多酚氧化酶活性显著高于海南样地，而尿酶活性、蔗糖酶活性远低于海南样地，磷酸酶和纤维素酶在两样地间无显著差异；5种内源激素含量在样地间和不同生长阶段植株间均差异；采用常规的TTC法检测孢子活力发现只有少量孢子染色，且染色时间较长，可能大量孢子失活；采用多种方法均未培养出蛇足石杉配子体和幼苗，但野在外采集到蛇足石杉配子体，了解了配子体形状，基本弄清了蛇足石杉孢子萌发过程和生活史。通过本项目实施发表论文2篇，投稿SCI论文2篇，撰写论文2篇，申请专利1项。