拟穴青蟹生殖免疫相关的抗菌肽互作蛋白ScyIP的特性及其互作机制研究

SCY2 is a novel antimicrobial peptide that is first identified in Chinese important economic mariculture breed S.paramamosain and proves to exert a main role in reproductive immunology. During the study, a novel uncharacterized protein named as ScyIP was discovered in Scylla paramamosain, which was an interacting protein of SCY2. This protein was highly expressed in testis of male S. paramamosain and possessed potent antimicrobial activity and apoptotic activity. In view of the correlation between SCY2 and ScyIP, it would be expected that ScyIP was likely to play a synergistic or combined action with SCY2 in the reproductive immunity but the underlying mechanism remains unknown and needs further investigation. As it is known, the reproductive immunity is critical for guaranteeing parent quality and embryonic development of mud crabs. Based on the previous studies, the main purpose of our present project was: (1) to investigate on the transcript distribution and expression patterns of ScyIP in tissues of S.paramamosain under natural and various stimulation conditions; (2) to uncover the interaction domains of ScyIP and SCY2 using mammalian two hybrid system; (3) to reveal the subcellular location of ScyIP and its pathway involved in apoptosis; (5) to understand whether ScyIP could affect testis development and spermatogenesis by way of inducing apoptosis of target cells, thus elucidating the fundamental features and functions of ScyIP; (6) Furthermore, try to clearly understand whether ScyIP participated in or partly joined with SCY2 in the reproductive immune defense through the studies on their synergistic antimicrobial activities of ScyIP and SCY2 and the influence of the changing hormone level on ScyIP expression. Finally, this project will provide scientific support for facilitating elucidation of the underlying mechanisms of an anionic antimicrobial peptide, which would be a basic guideline for maintaining the parent quality and embryonic development of S. paramamosain healthy while living in the complex environment.

SCY2是在我国重要海水养殖拟穴青蟹（简称青蟹）中发现的一种新型抗菌肽，具有生殖免疫功能。前期研究中发现一个与SCY2互作的新蛋白ScyIP，ScyIP在雄蟹精巢中高表达，抗菌活性强且可致细胞凋亡，推测其在生殖免疫中具有重要协同作用，生殖免疫对于保障青蟹亲体质量和胚胎发育等具有重要意义。本项目拟在此基础上，确定ScyIP的体内分布、刺激诱导表达特性及其抗微生物作用；开展ScyIP细胞凋亡途径的研究，揭示其是否通过引起靶细胞凋亡影响青蟹精巢发育及精子生成过程；从而初步阐明ScyIP的基本特性与功能。通过研究ScyIP和SCY2的联合抗菌作用，进行亚细胞定位及确定ScyIP与SCY2的互作位点，以及测定激素水平变化对ScyIP表达的影响等互作机制，揭示ScyIP与SCY2是否协同或联合发挥生殖免疫功能。该成果将为深入研究阴离子抗菌肽作用机理、保障青蟹亲体质量和胚胎发育健康等提供重要科学依据。

本项目以拟穴青蟹为研究对象，基于实验室前期发现的青蟹雄性性腺特异表达抗菌肽SCY2，通过酵母双杂交技术筛选获得其互作蛋白ScyIP，将新发现的该互作蛋白命名为Scyreprocin。本研究探讨了青蟹抗菌肽SCY2互作蛋白Scyreprocin的基本特性与功能, Scyreprocin与SCY2的互作机制及其在精巢发育及精子发生过程中的生殖免疫相关作用等。重要成果如下：①首次筛选获得青蟹抗菌肽SCY2的一种新型互作蛋白Scyreprocin，揭示了其基因结构，阐明其在自然状态及不同生理条件下的体内表达特性，发现该基因及蛋白在性成熟雄性青蟹性腺特异性表达，精巢中表达水平最高。②发现Scyreprocin与SCY2共定位于成熟精子的内质网、高尔基体及线粒体中，在精子顶体帽上亦存在共定位；③获得了Scyreprocin重组蛋白，该表达产物具有良好的抗菌活性，抗菌谱较广，能够有效杀伤细菌、真菌、抑制霉菌孢子发生、破坏霉菌菌丝结构等；rScyreprocin主要通过其自身的正电荷与细菌膜表面结合，改变细胞膜通透性、甚至直接对细胞膜结构进行破坏，导致细菌活力下降及死亡；能够引起真菌产生细胞凋亡效应。此外，rScyreprocin对感染哈氏弧菌的海水青鳉具有显著的免疫保护作用，能够显著提高青鳉的存活率；④rScyreprocin与rSCY2对真菌生物膜的形成有抑制活性，且具有联合或协同作用，rSCY2能增强rScyreprocin的抗生物膜活性；⑤首次揭示两种抗菌肽Scyreprocin与SCY2是青蟹精子顶体反应的关键因子，在孕酮的介导下，存在于精子顶体帽上的Scyreprocin作为精子孕酮膜受体与孕酮结合，参与了青蟹授精过程中的关键步骤。本项目为深入揭示Scyreprocin及SCY2的体内外作用机制及其在畜牧水产养殖业、医药行业等领域的应用奠定了基础。