摘 要

丹顶鹤（Grus japonensis），大型涉禽，我国Ⅰ级保护动物，CITES附录Ⅰ级保护动物，IUCN红皮书濒危物种，现今野生丹顶鹤种群数量约2800只，一为大陆迁徙种群，另一为日本驻留种群。近年来由于人口的增长，人类活动面积扩大，丹顶鹤栖息空间丧失。且人为的活动也使得丹顶鹤繁育能力以及成活率降低。

主要组织相容性复合体(MHC)是脊椎动物基因组中紧密连锁的一个基因家族。其遗传变异水平与动物的疾病抵抗能力密切相关，直接影响个体乃至整个种群的生存适应能力。本次实验对保护区内圈养丹顶鹤的MHC基因进行分离克隆，希望能为丹顶鹤的保护提供帮助。主要结果为：

1. 以鹤类动物MHC-I基因高变区通用引物设计得到丹顶鹤基因组DNA，最终分离得到了3条丹顶鹤I类MHC基因序列，且3条基因序列都具有能发生突变的可变位点，且都具有正常的编码蛋白质的功能。
2. 在得到的有效长度为543bp的序列中，含有16个可变位点，527个保守位点。分析之后得到序列中碱基组成符合碱基配对原则。3条序列氨基酸的差异度为5.52%，可变位点与序列长度的差异度为2.90%。
3. 利用ML算法和NJ算法得到的系统发育树显示不同参考物种MHC基因的进化方向与物种之间的进化方向是相一致的。

关键词：丹顶鹤，Ⅰ类MHC，跨物种进化，遗传多样性。

Cloning and Isolation of Class I MHC Gene from Grus

japonensis

ABSTRACT

Grus japonensis, large waders, China Class I protected animals,CITES Appendix I protected animals, and IUCN Red Book endangered species. The current population of wild red crowned cranes is approximately 2800, divided into two major populations, one is the mainland migratory population, and the other one is the population who always live in Japan. In recent years, because of population growth and the area of human activities has increased ,the habitat of red-crowned cranes has been lost so much. Then man-made activities also reduse the red-crowned crane breed and the survival rate.

The Major Histocompatibility Complex (MHC) is a family of genes closely linked in the vertebrate genome. The level of genetic variation is closely related to the animal's disease resistance, which directly affecting the individual's or even the entire population's ability to adapt uncultivated environment. This experiment was designed to study the isolation and cloning of MHC genes of captive cranes in protected areas, hoping to provide assistance for protecting red-crowned cranes.

1. The genomic DNA of red-crowned cranes was obtained by using universal primers for the hypervariable regions of the MHC-I gene of cranes,and three MHC sequences were found., all of them have normal protein-coding functions.
2. Obtaining sequence with an effective length of 543 bp, there are 16 variable sites and 527 conserved sites. The base composition in the sequence is in accordance with the principle of base pairing. And the amino acid diversity of the three sequences was 5.52%, the degree of difference between the variable site and the sequence length was 2.90%.

3)Phylogenetic tree obtained by ML algorithm and NJ algorithm showed that the evolution direction of MHC gene of different reference species was consistent with the evolutionary direction of species.

Key words：red-Crowned Crane,MHC class Ⅰgenes,Isolation and cloning,Genetic diversity.

目 录

1.前言 1

1.1丹顶鹤简介 1

1.1.1丹顶鹤生物学特征 1

1.1.2丹顶鹤保护现状 1

1.2MHC简介 2

1.2.1MHC结构及其分类 2

1.2.2MHC研究现状 3

1.2.3MHC在保护生物学的应用 3

1.3研究内容、目的及意义 4

2.丹顶鹤MHC-I的克隆与分离 5

2.1实验材料 5

2.1.1实验样品 5

2.1.2实验试剂 5

2.1.3实验仪器 5

2.2试验方法和步骤 5

2.2.1样品的采集与保存 5

2.2.2血液基因组DNA的提取 6

2.2.3PCR扩增反应 6

2.2.3电泳检测回收 7

2.2.4分子克隆 8

3结果 9

3.1丹顶鹤MHC-I部分扩增结果 9

3.2碱基组成分析 10

3.3氨基酸对比分析 10

3.3.1氨基酸序列对比分析 11

3.3.2氨基酸成分比较分析 11

3.4系统发生分析结果 12

4结论 14

4.1本实验的主要结论 14

致谢 15

参考文献： 17

1.前言

1.1丹顶鹤简介

1.1.1丹顶鹤生物学特征

丹顶鹤（Grus japonensis），属大型涉禽，又称之为仙鹤、红冠鹤，在中国的传统文化中也具有清廉公正、品德高尚的寓意。是我国Ⅰ级保护动物，CITES附录Ⅰ中保护动物，2000年被IUCN红皮书列为濒危物种¹。丹顶鹤是鹤科鹤属的物种，体长120～160厘米，雌性丹顶鹤身形体重略大于雄性丹顶鹤。丹顶鹤脖颈、下肢较长，身形看上去高挑。丹顶鹤身躯被羽毛，大多雪白，耳后至头枕白色，其头顶无羽毛包被，鲜红色，颊、喉、脖颈、下肢黑色²。尾部的次级飞羽和三级飞羽为黑色，且三级飞羽较长弯曲形似弓状³。嘴较长，突出呈现灰绿色，尖端为黄色。未成年的丹顶鹤被有绒羽，3～4月大后绒羽逐渐由黄褐色变为雪白，头顶显现为红色的裸露部分则要到10个月以后才能看出³。

丹顶鹤平常常以小群的小家族形式群居于有芦苇的浅水地带，主要为苇草滩和盐蒿滩等⁴。如有较多丹顶鹤个体集体活动的种群出现于农田和耕地中时，则为丹顶鹤的迁徙季节²。迁徙时节分为繁殖迁徙或过冬迁徙，2月末或3月初时的为离开越冬地迁往繁殖地进行繁殖活动的迁徙；9月末或10月初时则为离开繁殖地往南的越冬迁徙³。丹顶鹤每窝产卵1～2枚，卵为椭圆形，苍灰色或灰白色，孵化期通常为30～33天。