黑斑侧褶蛙抗菌肽进化与微生物群落关系的研究

Antimicrobial peptide (AMP) is the major innate immunity component to protect amphibians from being attacked by environmental microbes. Therefore, the evolution of AMPs would be influenced by microbial community. Some molecular evolution mechanisms of AMPs have been discovered in recent years, such as focal mutation, gene duplication, domain shuffling, divergent selection and balanced selection etc. However, there has been very limited study on the association of AMP evolution and microbial community, leaving these molecular mechanisms lack of ecological evidence. In order to study the association, this project takes the black-spotted frog, a wide spread species with clear dispersal history, as material, samples frog skin and soil as well as water microbes from its refugium during the latest glacier period and six dispersal ranges, analyses microbial community structure and component of AMP of each sampling site, and proves three hypothesis: 1) there are two strategies adopted by AMPs, the specific strategy in cope with novel pathogens and the spectrum-broadening strategy in favor of local enhancement of defense spectrum; 2) spectrum-broadening strategy makes alterations of AMPs independent of microbial community, masking the association between AMP and microbial community; 3) The geographic pattern of AMPs is shaped by the relative intensity of the two strategies rather than the microbial community.

两栖动物用皮肤分泌的抗菌肽来抵御环境中微生物的侵袭，因而抗菌肽的进化应与微生物群落之间存在必然联系。近年来已从分子水平上陆续揭示了抗菌肽的进化机制，如快速点突变、基因复制、结构域混编、歧化选择与平衡选择等，但是与微生物群落之间的关系还没有系统研究，导致这些机制缺乏生态学上的印证。本项目以种群扩散历史清楚的广布种黑斑侧褶蛙为材料，拟从其冰期庇难所和6个扩散地采集皮肤和土壤表层、水体样品，通过第二代测序技术分析各地区细菌和真菌群落的结构及抗菌肽组成，基于系统发育关系分析微生物群落进化与抗菌肽进化的关系，验证3个科学假设：1）抗菌肽以广谱性和特异性两种策略进化，特异性策略用于应对新抗原，而广谱性策略用于抗菌谱的局部扩大和加固；2）广谱性策略使抗菌肽的变化不依赖于微生物群落成员的变化，从而导致二者之间的协同关系变得模糊；3）两种策略的相互消长主导抗菌肽地理格局形成。

两栖动物皮肤主要靠抗菌肽来防御环境微生物的攻击。抗菌肽由众多基因家族编码，受到正选择的驱动形成极高的多样性。但是，在抗菌肽系统形成过程中，如果每个基因都受到正选择作用，那么该系统就会十分纷乱，耗费极多的进化成本。本项目以黑斑侧褶蛙为模型，以抗菌肽基因结构、选择方式、转录子组成、与环境微生物的关系等入手，系统研究抗菌肽的进化方式。结果显示，以Temporin为代表的抗菌肽抗菌谱极为广泛，可参与很多生理功能，但基因十分保守，具有多座位、高表达的特点，受到强烈的负选择压力；而以Esculentin为代表的抗菌肽则多样性很高，进化受到正选择驱动。抗菌肽的整体表达谱与环境微生物群落的关系模糊，特定抗菌肽的表达量与特定微生物类群可能存在对应关系。这些结果支持了我们的推测：1）抗菌肽以广谱性和特异性两种策略进化，特异性策略用于应对新抗原，而广谱性策略用于抗菌谱的局部扩大和加固；2）广谱性策略使抗菌肽的变化不依赖于微生物群落成员的变化，从而导致二者之间的协同关系变得模糊；3）两种策略的相互消长主导抗菌肽防御系统的最终结构。