水蛭长期储藏食物策略的机制研究

Hirudo nipponica is the main raw animal of medicinal, which is one of the main raw meterials for a variety of proprietary Chinese medicine treatment of cardiovascular disease. The leech has long-term in vivo storage of food survival strategies. At present, the relevant reports are leech food storage mechanism. This paper intends to study the leech and crop food storage at different feeding stages and changes in the law, to find the difference of bioactive substances and screening of clear crop main symbiotic microorganisms; followed by separation and purification with antibacterial function of leech crop and food in the protein, determination of its amino acid sequence and clearly isolated according to classification; protein and combining existing transcriptome data, and cloning of genes related to antibacterial function. Comprehensive comparison and analysis of the food intake before and after difference material, purified antibacterial protein gene and molecular cloning of the corresponding protein, screening three mutually matched protein with antibacterial protein, and study the function; finally through space-time determination of antibacterial gene expression differences, clarify its mechanism, constructing different balance model thus, revealing the action mechanism of antimicrobial protein in the crop and the mechanism of long-term storage of leech crop. Meanwhile, the study can provide a new theoretical basis for the research on animal food storage behavior.

水蛭Hirudo nipponica为药材水蛭的基原动物之一，是治疗心血管疾病中成药的主要原料，水蛭具有长期体内储藏食物的生存策略，目前有关水蛭食物储藏机理鲜见报道。本课题首先拟通过研究水蛭嗉囊及其储存食物在不同摄食阶段的差异及变化规律，寻找差异生物活性物质，并筛选明确嗉囊内主要共生微生物；其次分离纯化水蛭嗉囊及食物中具有抑菌功效的蛋白，测定其氨基酸序列并明确分类；根据分离纯化获得的蛋白并结合已有转录组数据，并克隆抑菌功能相关基因。综合对比分析筛选具有抑菌活性蛋白，并进行功能研究；最后分别通过测定相关抑菌基因的时空表达差异，阐明其作用途径，构建不同的平衡模式，从而揭示上述抑菌蛋白嗉囊内的作用机制，明确水蛭嗉囊长期储藏这一生存策略的机理研究，同时可为动物贮食行为研究提供新的理论基础。

日本医蛭为药材水蛭的基原动物之一，以动物新鲜血液为食，具有抗凝、抗血栓等功效，是常用活血化瘀药及心血管疾病中成药的主要原料 。本课题首先通过研究水蛭肠道菌群在不同摄食阶段的差异及变化规律，明确Mucinivorans、Elstera和气单胞菌为水蛭肠道内主要共生菌，发现水蛭长期储食与其肠道共生菌群功能有直接联系。结合水蛭摄食过程转录组数据，发现免疫、血管舒张、消化和发育等相关基因参与了日本医蛭摄食的过程；筛选出摄食过程中与抗菌相关的重要基因Hmc。透射电镜结果表明，水蛭抑菌蛋白Hmc体外通过裂解枯草芽孢杆菌及金黄葡萄球菌的细胞膜达到抑菌作用；Hmc蛋白定位于细胞核外，水蛭唾液腺中Hmc荧光强度在摄食中和摄食后阶段显著升高，并在摄食后期阶段达到峰值。日本医蛭在摄食期间体内抗氧化相关酶、免疫相关酶活性均呈现出先下降后上升的趋势，体内Hmc、免疫信号通路相关基因以及溶菌酶相关基因在摄食后均显著上调。进一步发现Hmc可以直接减少野生型秀丽隐杆线虫及pmk-1突变体线虫肠道外来病原菌的数目，提高了野生型线虫pmk-1/p38 MAPK途径关键基因（pmk-1、tir-1、atf-7、skn-1）以及抗菌效应物（clec-82、nlp-29、lys-1、abf-2）的表达，但未能延长和提高pmk-1突变体线虫寿命和抗菌效应物基因的表达， Western blot进一步证明，Hmc显著提升了野生型线虫体内pmk-1蛋白表达水平。水蛭长期储食的机制：水蛭肠道共生菌能与外源细菌竞争主导地位，快速平衡肠道菌群；同时水蛭Hmc蛋白是具有直接抑菌和调节先天免疫双重作用的蛋白，通过直接或调节水蛭免疫能力抵抗外源细菌，协助共生菌平衡肠道菌群，从而达到长期储食目的。