朱鹮MHC基因组解析及种群的环境适应性研究

Crested ibis is a first-class protected bird in China and also listed as endangered in the International Union for Conservation of Nature (IUCN). The crested ibis was once distributed widely in China but habitat destruction and serious poaching caused that only seven birds survived in the Qinling mountains in 1981. Since then, Chinese government has taken great efforts to save this speices and has expanded the ex- and in-situ populations to current 1700 birds. Although the crested ibis has revived from the brink of extinction, the populations have exhibited high rates of pathogen infection and mortality as the extent of inbreeding has been going up. On the other hand, the major histocompatibility complex (MHC), which encodes molecules that bind short peptides from pathogens, present them to immune cells and trigger an immune response, plays a central role in the vertebrate immune system. The MHC genes form a well-known multiple-locus function system; the number of its loci decides the ability of antigen-presentation and the polymorphism of respecitve locus determines the sorts of pathogen-derived antigens. Since (1) asymmetry of multi-locus MHC haplotypes is a common phenomenia in the birds and (2) inbreeding leads to reduction of polymorphism and unmasking of deleterious recessive alleles, extreme inbreeding would bring double risks to the bird MHC system, that is simultaneous loss of alleles and loci. As a result, this project intends to (1)fully determine the MHC genome of the crested ibis using a new reverse-4D method; (2) develop genotyping technique of MHC genes to investigate adaptive variability ofcrested ibis populations; (3) examine the relation between pathogen susceptibility or mortality rate and MHC polymorphism; (4) make conservation recommendations from the perspective of functional immune genes to ensure the long-term survival of the crested ibis.

朱鹮是我国一级保护的濒危鸟类，于1981年经历了仅剩7只野生个体的严重瓶颈！目前我国已恢复到1700多只朱鹮，但严重的近亲繁殖导致了逐年上升的病原感染率和死亡率！MHC是负责抗原呈递的免疫"开关"基因，它的位点数量决定着抗原呈递的能力，位点的多态性决定着其呈递的抗原种类多少！极度近交意味着基因的高度纯合，那么朱鹮的病原易感是否与近交导致的MHC免疫能力下降有关呢？极度近交同时意味着隐性有害基因的暴露，那么朱鹮的高死亡率是否与近交导致的免疫缺陷有关呢？在朱鹮已经脱离灭绝边缘的今天，我们该如何从免疫功能基因的角度提出合理有效的保护策略，以确保朱鹮的长期生存呢？鉴于此，本项目拟利用前期建立的反4D技术和相关的生物信息分析手段解析朱鹮的MHC基因组，建立全套MHC位点的分型技术，查明朱鹮的环境适应能力现状，阐明朱鹮高死亡率与MHC基因抗病能力缺失的相关性，最终为制定行之有效的保护策略提供科学依据！

朱鹮（Nipponia nippon）作为世界濒危物种及国家一级重点保护动物，曾一度濒临灭绝。1981年在中国陕西省洋县重新发现的7只野生朱鹮是目前所有已知个体的奠基者。如今种群数量已突破2000只，但由于曾经历严重的瓶颈效应及近亲交配，其种群在变幻莫测的环境压力下仍危机重重，发展前景依旧未卜。..MHC是脊椎动物免疫系统中高度多态的一种多基因家族，负责识别并传递抗原，并启动免疫应答，是一种热门的适应性分子标记。本研究欲构建朱鹮的BAC基因组文库，解析朱鹮MHC基因组，研发MHC分子标记系统，最后对圈养种群和野生种群进行分子检测，阐明朱鹮种群的遗传适应威胁，并提出科学有效的保护策略。.本研究取得的主要结果如下：..（1）构建了7.65倍朱鹮基因组覆盖度的传统4D文库和约40倍覆盖度的新型反4D文库，从而获得单拷贝基因的概率约为100%。该组合文库的实用有效性通过解析150kb长的先天性免疫防御素基因区段得到证明；..（2）基于BAC文库，解析了长达500kb的朱鹮MHC基因组。BAC测序结果表明，朱鹮的MHC具有多个αβ串联重复的独特特征，且首次证明鸟类祖先具有两套差异巨大的αβ二聚体，对鸟类MHC演化史的认知产生了革命性影响；..（3）研发了朱鹮先位点特异性分型和再长片段连锁分型的分子标记系统，分型结果表明朱鹮的MHC经典区共存在7个II-I类连锁单倍型，又可分为四组：HT01（含4个“αβ”）、HT02与06（含3个“αβ”）、HT03与07（含2个“αβ”）、HT04与05（含1个“αβ”）。朱鹮这种位点不等现象说明其潜藏MHC缺陷的致死威胁，圈养种群需要人工干预配对计划，提高种群生存力；..（4）圈养种群的MHC基因分型与子代死亡的相关性分析表明，“αβ”单元的缺失与子代高死亡率有直接关系，且以基因型HT03/HT04与子代死亡最相关。该结果说明，人工配对计划中应避免HT03/HT04基因型的出现，以提高子代生存率；..（5）朱鹮的群体检测结果表明，其当前的遗传多样性水平较低，遗传漂变是朱鹮的主要进化动力。不同种群在不同标记上却体现出一致的遗传分化，且多方面证据表明朱鹮存在多样化选择，有利于对抗遗传漂变造成的多样性丢失，维持物种生存力。