【作者】 李晓平；

【导师】 徐艳春；

【作者基本信息】 东北林业大学 ， 特种经济动物饲养， 2010， 硕士

【摘要】 抗菌肽具有广谱杀菌、抑制病毒、抑制肿瘤细胞等作用,是两栖类动物天然免疫的主要成分。不同的环境中,抗原组成不同,两栖类机体会选择性地表达不同的抗菌肽谱。因而抗原是抗菌肽进化的主要压力。动物体如何根据环境抗原来变化表达谱呢?目前关于这个问题尚无研究可鉴。针对这个问题,我们2009年6月份在哈尔滨市郊采集黑斑蛙(Rana nigromaculata)作为研究对象,一组在野外捕获后立即处死,采集皮肤样品,另一组在实验室内用清水饲养3周后再处死,采集皮肤样品。通过比较两种情况下抗菌肽谱的异同点,尤其是抗菌肽的种类、理化性质、选择模式等,来阐释不同环境下抗菌肽的表达模式。结果显示,黑斑蛙共表达10个家族的22种抗菌肽,由65个基因编码的71种cDNA组成。其中有7个家族的17种抗菌肽(62种cDNA,分属于esculentin-2家族、nigrocin-2家族、odorranoin-S1家族、temporin-1家族、pelophylaxin-2家族、odorranain-P1家族,odorranain-B1家族)是实验室组和野外组所共同表达的,而有2个家族的4种抗菌肽(8种cDNA分属brevinin-1家族和pelphylaxin-3家族)为野外组所特有,1个家族的1种抗菌肽(3种cDNA属于nigromaculatin家族)为实验室组所特有。两组共有的家族和野外组特有的家族中,都属于碱性抗菌肽,除了nigrocin-2家族和odorranoin-S1家族以自由卷曲结构为主以外,其余家族的抗菌肽均形成轴向和断面两个维度上具有两亲性的α-螺旋结构。实验室组特有的抗菌肽nigromaculatin家族属于自由卷曲的酸性抗菌肽。temporin-1家族的cDNA达22种,且总表达量最高,为较强烈的负选择抗菌肽,野外组表达量稍高于实验室组。esculentin-2家族的cDNA有16种,但总表达量不高,亦为负选择或近于中性选择的抗菌肽。pelphylaxin-3家族包含6种不同的cDNA,在野外组总表达量仅次于temporin-1家族,均为正选择抗菌肽。odorranain-B1家族、brevinin-1、nigromaculatin家族的表达量均很低,而且都属于负选择抗菌肽。统计表明,随着与祖先基因的遗传距离的增加,抗菌肽表现出从负选择逐渐过渡到正选择的倾向。上述结果提示,(1)黑斑蛙基因组上至少具有65个基因编码71种抗菌肽。(2)在不同环境中黑斑蛙抗菌肽表达谱中有稳定的组分和环境特异性组分。稳定组分中的抗菌肽基因比较古老而保守,环境特异性组分的抗菌肽基因通常出现较晚,且以正选择模式进化。(3)环境抗原较少时(如实验室组),抗菌肽的表达从种类和丰度上均明显减少,即抗菌肽的表达具有明显的经济性原则。更多还原

【Abstract】 Antimicrobial peptides (AMPs) are an important component of innate immunity of amphibians due to their functions of broad spectrum of bactericide, viral and tumor resistance. Amphibians use different AMP profile selectively to cope with different pathogenic community in different environment. This indicates an essential role of pathogenic community in the evolution of AMPs and raises a question how amphibians choose AMP profiles in coping with different pathogenic community. There has not been definite answer to this question up to date. We analyzed AMP profiles of black-spotted frog (Rana nigromaculata) in June 2009 in Harbin, China. Adult frogs were captured in the wild and divided into two groups randomly. One group was killed in situ immediately and skin samples were collected and preserved in liquid nitrogen. The other group was transferred into the lab and sampled skins after being raised in clear water for three weeks. Profiles, physical and chemical properties and mode of selection of AMPs were compared between the two groups. Seventy one cDNAs from 65 genes encoding 22 species of AMPs belonging to 10 families were cloned in total. Seven families containing 17 species of AMPs encoded by 62 cDNAs belonging to esculentin-2, nigrocin-2, odorranoin-S1, temporin-1, pelophylaxin-2, odorranain-P1 and odorranain-B1 were shared by the two groups. Two families of 4 species of AMPs encoded by 8 cDNAs belonging to brevinin-1 and pelphylaxin-3 were unique to the wild group. One species of AMP encoded by 3 cDNAs that was identified in ranids for the first time and named nigromaculatin was only seen in the lab group. All shared AMPs were alkaline and cationic peptides with typical a-helix structure except for family nigrocin-2 and odorranoin-S 1 with random coil structure. Family nigromaculatin was acidic and randomly coiled AMP. Family temporin-1 contained 22 cDNAs evolving under negative selection and revealed the highest expression, a little higher in the wild group relative to the lab group. Family esculentin-2 contained 16 cDNAs evolving under negative selection as well or near neutral selection. But their expression was moderate. Family pelphylaxin-3 containing 6 cDNAs under positive selection was the second abundant AMPs in the wild group relative to family temporin-1. Family odorranain-B1, brevinin-1 and nigromaculatin were all under negative selection but with low expression. Analysis also indicated that the selection of AMPs was in a trend of changing from negative selection to positive selection with increment of genetic distance relative to their ancestral genes. These results suggested that (1) the black-spotted frog has at least 65 gene loci encoding AMPs; (2) there are stable components and environmental specific components in AMP profiles when the black-spotted frog is exposed to different environment. The genes of stable AMP components are primordial and conservative. Whereas, the genes of environmental specific AMP components are usually recent and evolving in positive selection; (3) In case of simplification and reduction of pathogenic community such as lab group was, AMP expression may reduce in both AMP species and their abundance, indicating the expression of AMPs follow economic principle.更多还原