【作者】 陈礼诚；

【导师】 方盛国； 万秋红；

【作者基本信息】 浙江大学 ， 生态学， 2013， 博士

【摘要】 朱鹮(crested ibis, Nipponia nippon)是我国一级保护鸟类,也是世界濒危动物保护的旗舰物种之一。该物种自上世纪中后叶一度近乎灭绝以来,经过30多年的保护和繁育,已在个体数目上有了较显著的增长。但由于经历过极其严重的瓶颈,朱鹮种内的遗传多样性仍然很低,其种群在病原体等环境压力之下仍显脆弱,因此还并未完全摆脱灭绝的危险。主要组织相容性复合体(Major Histocompatibility Complex, MHC)是脊椎动物免疫系统的重要组成部分,对动物抵抗病原体、适应外界环境至关重要。不仅如此,MHC还在动物的亲缘识别、配偶选择等方面扮演着重要角色,与物种的繁衍也息息相关。因此,MHC分子的结构及功能、多态性及其维持机制、在性选择中发挥的作用及机制,等等,为保护生物学、免疫遗传学等诸多领域的学者所关注。而通过构建基因组细菌人工染色体(Bacterial Artificial Chromosome, BAC)文库,利用BAC来获得目标物种MHC的详细序列信息,是研究MHC的有效方法。基于BAC构建MHC详细的物理图谱,获得MHC区域内基因位置和序列信息,有助于全面而准确地分析物种MHC的特征,以及种间MHC的进化关系。综上原因,本研究构建了覆盖7.65倍基因组大小的具备4D-PCR筛选系统的朱鹮基因组BAC文库；同时还用Reverse-4D方法构建了基因组覆盖率更大的BAC文库,以作为前者的补充,并更好地保存朱鹮的基因组DNA资源。在MHC阳性BAC筛选及其测序的基础上,本研究构建了到目前为止首份鹮科鸟类的MHC基因组精细物理图谱,获得了朱鹮MHC区域的详细碱基序列。该图谱主要分为MHC核心区、BG区以及Ⅰ类区三大块,共约475kb。尽管上述三块MHC区域的位置关系仍待后续工作检验,但在其上已共预测到了包括MHC Ⅰ类、Ⅱα、Ⅱβ、DM、TAP、BG等基因在内的50个基因位点。朱鹮MHC在展现出与鸡等鸟类MHC结构大致相似的同时,更具有物种本身的特征。特别是MHC核心区Ⅱ类区域中存在相邻的α和β位点,提示了鸟类MHC结构的多种进化方向。目前朱鹮已知的MHC Ⅰ、Ⅱ类基因位点数以及MHC覆盖的区域大小,也进一步提示鸟类中存在的"minimal essential MHC"可能只属于鸡形目等少数鸟类,更多的鸟类MHC特征信息仍有待研究挖掘。由于鸟纲物种分布广泛,分类庞杂,且MHC在不同支系的鸟类中表现出多样的进化特征,因此需要有更多不同目乃至更低分类级的鸟类物种MHC的信息,才能全面而清晰地呈现出鸟类MHC分化及各自演化的历程。而本文的研究,使上述工作又向前迈出了一小步。同时,朱鹮MHC精细物理图谱的构建,以及详细MHC基因序列的获得,为今后以MHC为研究对象和切入点的朱鹮物种保护及繁育工作,提供了坚实基础。更多还原

【Abstract】 The Crested ibis (Nipponia nippon) is a national first-class protected species in china, as well as one of the most endangered bird species in the world. It was almost extinct in the last century. Fortunately, after30-years protection, the number of individuals of ibis has increased significantly. Despite the expansion in population, this species is still vunerable to pathogens and other environment stresses and under the threat of being extinct, due to its low immune genetic variety caused by the severe bottleneck it had suffered. The major histocompatiility complex (MHC) is of great importance in vertebrate immune system, playing roles in the resistance to pathogens and adaption to environment. MHC also takes part in the recognition of relationships and mate choice, which is closely related to reproduction. Therefore, MHC is paid attention to and being studied by many scientists in different fields such as conservation biology and immunol genetics. It is effective and efficient to study MHC based on construction of bacterial artificial chromosome library and sequencing of BACs containing target MHC sequences. Since MHC contains lots of gene loci, it is necessary to acquire detailed and integrate sequence based on a physical map of MHC, for the purpose of learning MHC in an overall view.In this research, we constructed a crested ibis BAC library with4D-PCR screening system, providing about7.65-fold coverage of the crested ibis genome; we also generated an additional BAC library which provides even more coverage of the genome by using reverse-4D process. Based on the screening and sequencing of MHC positive BACs, we achieved, to our knowledge, the first physical map of MHC in Threskiornithidae birds, as well as sequences that totally span about475kb of crested ibis MHC. The ibis MHC is composed of three regions, the MHC core region, the BG region and the class Ⅰ region. It is still unknown whether the three parts of MHC were located on the same chromosome and closely linked to each other. A subsequent study to resolve this question will be done soon. Nevertheless,50gene loci have already been predicted and defined in the ibis MHC, including MHC Ⅰ、Ⅱα、Ⅱβ、 DM、TAP、BG and so on. While showing some similarity on the organization of MHC genes to that of chicken, the ibis MHC displays different and specific features of itself. The most notable difference between ibis and chicken MHC maybe the apparence of Ⅱα loci located next to Ⅱβ loci in the ibis core MHC region, which indicates a variety of evolution directions of MHC structure in birds. Compared to chicken and turkey MHC, the ibis MHC spans a much extended region with more MHC class Ⅰ and Ⅱ loci, suggesting that the "minimal essential MHC" may be limited in only a few bird orders, and more informations of avian MHC are waiting to be unveiled.更多还原