肉垂鹤I类MHC基因的克隆分离研究毕业论文

 2021-04-20 11:04

摘 要

肉垂鹤(Bugeranus carunculatus)是非洲陆地上所特有的物种,到目前为止只有8000只存活,而肉垂鹤所赖以生存的地区,湿地等目前被大量开发,加之小种群效应,肉垂鹤正在面临严重的灭绝旋涡,遗传多样性的减少也使得肉垂鹤在经历一些外界的疾病时,抵抗力显得尤为弱小。关于肉垂鹤微观分子方向研究非常少,所以本实验我们选择了主要组织相容性复合体(Major Histocompatibility Complex),它是脊椎动物免疫系统的一个非常重要的组成部分,由于自身基因序列排序的特点,显现出非常多变的多态性。实验研究结果如下:

1)本次研究从一只肉垂鹤的gDNA中分离出3条I类MHC基因序列。序包含部分外显子1到外显子4序列,共扩增总长度约1400bp, 裁掉内含子1,余下543bp的外显子2-4。这三条序列均能翻译成正常的氨基酸序列,其间无终止,证明分离得到的三条序列全部都来自经典的MHC基因座位。

2) 用Mega分析出三条序列里各个碱基组成的情况:三条Buca-UA中T(U)的平均含量为16.1%,C的平均含量为25.8%,A的平均含量为23.5%,G的平均含量为34.5%。具体见实验结果,三条序列碱基的平均含量T(U)含量较少,G的含量最多。

3)根据所构建的系统发育树,Buca-UA-01和Buca-UA-02以及Buca-UA-03形成独立的进化枝,所以跨物种进化也是肉垂鹤的I类MHC基因的非常重要的进化模式。

关键词:肉垂鹤;MHC基因;位点分离;跨物种进化

Cloning and isolation of MHC gene from cranes

ABSTRACT

Caruncle crane (Bugeranus carunculatus) is endemic to Africa land species, with only 8000 so far, and caruncle cranes to the survival of the region, wetland is a large number of development, combined with small population effect, caruncle cranes are facing serious extinction vortex, reduce genetic diversity makes caruncle crane after some outside of disease, is particularly weak resistance. Caruncle crane micro molecular direction of research is very little, so this experiment, we chose the Major Histocompatibility Complex (Major Histocompatibility Complex), it is the vertebrate immune system is a very important part of due to the characteristics of gene sequence sorting itself, appear very changeable polymorphism. The experimental results are as follows:

1) In this study, three sequences of I MHC genes were isolated from the gDNA of a crane. The sequence contains partial exons 1 to exon 4 sequences, total amplification of the total length of about 1400bp, cutting intron 1, and the remaining 543bp exon 2-4. These three sequences can be translated into normal amino acid sequence without termination, and the three sequences obtained from the separation are all derived from the classical MHC gene seats.

2) The composition of each base in three sequences was analyzed by Mega: the average content of T(U) in three buca-ua sequences was 16.1%, the average content of C was 25.8%, the average content of A was 23.5%, and the average content of G was 34.5%. As shown in the experimental results, the average content of T(U) of the three sequence bases is small, and the content of G is the highest.

3) According to the established phylogenetic tree, buca-ua-01 and buca-ua-02 and buca-ua-03 formed independent evolutionary branches, so the genetic recombination was also a very important evolutionary pattern of the type I MHC gene of the succulent.

Key words:Bugeranus carunculatus;MHC gene;Site separation;Cross species evolution

目 录

1 前言 1

1.1 肉垂鹤简介 1

1.2 关于肉垂鹤的国内外研究现状 1

1.3 主要组织相容性复合体基因简介 1

1.4 主要组织相容性复合体基因分类结构及分布 2

1.5 主要组织相容性复合体基因研究现状 2

2 实验内容 4

2.1 实验材料 4

2.1.1 实验样品和实验试剂 4

2.1.2 实验仪器 4

2.2 实验方法和步骤 4

2.2.1 血样的采集 4

2.2.2 肉垂鹤基因组的提取(基因组DNA提取试剂:DNAiso Reagent) 4

2.2.3 目的基因扩增 5

2.2.4 制胶和凝胶电泳 6

2.2.5 DNA纯化 6

2.2.6 连接与转化 7

2.2.7 送 测 8

2.2.8 生物信息学分析 8

3 结果与讨论 9

3.1 PCR扩增结果 9

3.2 菌液PCR扩增结果 9

3.3 碱基组成分析 9

3.4 氨基酸组成分析 9

3.5 系统发育树分析 10

结 论 13

致 谢 14

参考文献 15

1 前言

1.1 肉垂鹤简介

肉垂鹤(Bugeranus carunculatus)习惯于栖息在沼泽、浅滩、芦苇塘等湿地,以捕食小鱼虾、昆虫、蛙蚧、软体动物为主,也吃植物的根茎、种子、嫩芽。最近几年,因为湿地不断被开发破坏,肉垂鹤赖以生存的生境区域逐渐的减小甚至丧失,其生长分布区日益缩小,加上人为的猎杀以及大量流行疾病的蔓延,使得肉垂鹤的数量不断减少,加上物种小种群的危害。目前,非洲大陆上仅存8000只肉垂鹤,该物种已经被世界自然保护联盟(IUCN)列为濒危等级。濒危物种会表现遗传变异丧失、近交衰退、进化可塑性丧失等一系列问题,一旦种群状况不断恶化,该物种将卷入灭绝漩涡,最坏的结果就是从地球上消失,那么一系列的生态链断裂,后果不堪设想。

1.2 关于肉垂鹤的国内外研究现状

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