药用水蛭抗凝基因家族系统演化关系及其功能研究

水蛭的抗凝功能在防治心脑血管疾病方面具有特效作用，但不同水蛭抗凝效果及药材质量标准差异显著，导致使用过程中资源的浪费，亟需开展其抗凝功能的研究。本项目在前期工作基础上，筛选蚂蟥W. pigra、水蛭H. nipponica、菲牛蛭P. manillensis抗凝基因EST序列；采用RACE法分别克隆其相关抗凝基因；通过抗凝基因系统演化关系明确抗凝关键基因，采用荧光实时定量PCR、蛋白质印迹法测定关键基因以及蛋白的器官和吸食阶段特异性表达，通过测定抗凝血酶活性，构建抗凝基因表达与抗凝血酶活性关系图谱，探讨药用水蛭抗凝基因最佳时空表达模式。在此基础上，通过RNAi技术重点研究抗凝基因功能，最后构建分子系统进化树，实现药用水蛭抗凝基因家族系统演化关系及功能研究的目标。本研究对揭示药用水蛭抗凝基因功能和机制的研究具有重要科学意义，同时可为水蛭资源保护、人工养殖和科学制定药材质量标准提供理论依据。

The anticoagulant effect of Hirudo acts special role in preventing and treating cardiovascular and cerebrovascular diseases, nevertheless the difference of the anticoagulant effect and the medicinal quality standard in various Hirudo is remarkable, lead to a waste of use Hirudo resources, it is urgent to studies on its anticoagulant function. It screened out the sequence of EST gene on Hirudo anticoagulant, basing on the existing transcriptome sequence of W. pigra, H. nipponica, and P. manillensis and cloning the gene of anticoagulant through rapid amplification of cDNA ends (RACE) in this project. Aimed at the key anticoagulant gene by research on the evolution of the relationship between anticoagulant gene system, detecting the expression patterns of different Hirudo key anticoagulant genes and protein in different taking stages and different organs by Realtime-PCR and Western blot, building the relationship spectrum of anticoagulant gene expression and anti-thrombin activity by detecting the antithrombin activity, exploring the optimal space-time on anticoagulant gene of Hirudo to verify the function of the gene expression pattern. Moreover, it is emphasizing to study on anticoagulation effect of key gene by using RNAi interference experiment, it will reveal the evolution system and the function of the anticoagulant gene family on Hirudo by molecular phylogenetic tree. The results of this research not only can clear molecular evolutionary mechanism of anticoagulant gene function in Hirudo, but also provide more theoretical basis for research for the protection of resources, artificial breeding, and scientific development of medicinal quality standard of Hirudo.