长江草鱼群体MHC Class Ⅱ B基因的克隆、表达及多态性分析

The Cloning, Expression and Polymorphism of MHC Class Ⅱ B Gene of Grass Carp (Ctenopharyngodon Idella) Populations of Yangtze River

作者: 专业:水产养殖 导师:曾令兵 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

Grass carp (Ctenopharyngodon idella), MHC, evolutionary pattern, genetic variation, expression

        主要组织相容性复合体(major histocompatibility complex, MHC)是在脊椎动物中发现的编码免疫球蛋白样受体的高度多态的基因群,并且其某些基因型与生物体抗病能力存在一定的关联关系。本研究以我国重要淡水养殖经济鱼类草鱼为实验材料,利用PCR、RT-PCR等技术研究了MHCⅡB基因的克隆、表达、等位基因的多态性以及等位基因的多态性与鱼体抗病力之间的关系,为筛选抗病相关MHCⅡB基因标记和建立基因标记辅助选育技术提供基础。(1)克隆及测序了草鱼长江3个群体的主要组织相容性复合体(MHC) ClassⅡB基因编码β1和β2区的第2和第3个外显子及两个外显子之间的内含子,分析了草鱼MHC的进化模式和种群遗传结构。结果共定义了34个等位基因,每条序列包括长为130-136bp的第2个外显子,长为218bp的第3个外显子以及长81-371bp的内含子。序列分析揭示,第2个外显子有106个核苷酸变异位点(78%)和40个氨基酸变异位点(88%),而第3个外显子有100个核苷酸变异位点(45%)和41个氨基酸变异位点(56%),β1变异要大于β2区。用邻接法(NJ)分别构建的β1和β2序列系统树均获得5个高支持率的谱系,结合序列变异特点和内含子长度,推测草鱼至少存在5个MHC ClassⅡ座位。分别计算β1的肽结合位点(PBR)、非肽结合位点及β2的非同义替换率(dN)和同义替换率(dS),结果显示PBR的dN/dS为2.03(p<0.05),非肽结合位点和p2则小于1,表明草鱼MHC受到歧化选择作用。根据不同群体中MHC等位基因分布频率的分子方差分析(FST=0.37%),长江草鱼群体间无遗传分化。(2)用嗜水气单胞菌感染健康草鱼,分别在感染5h、16h、40h、64h、88h、112h时收集草鱼各组织材料提取总RNA,另取健康草鱼组织作对照。以反转录合成的第一链cDNA为模板,应用RT-PCR半定量检测方法对草鱼感染嗜水气单胞菌前后的MHCⅡB基因组织表达特点进行了研究。结果表明,在正常草鱼组织中,除脑组织以外,MHCⅡB基因均扩增出约350bp的片段,MHCⅡB基因较强的表达于肾脏、心脏和肠中,中等程度表达于肝脏、脾脏和鳃中,在肌肉中表达最弱。嗜水气单胞菌感染后,在草鱼的肝脏、肾脏、心脏、肠和鳃组织中,感染初期MHCⅡB基因表达量都有所下降,而在感染88h的时候MHCⅡB基因表达量在所有时间点中达到最大,感染112h后表达量慢慢回落至正常值;其中肠组织在112h时,MHCⅡB基因表达水平依旧较高。脾脏在感染后,表达量总体处于上升趋势,88h时表达量达到最大值,112h后慢慢回落至正常值。
    The major histocompatibility complex(MHC) is a group of closely linked loci presented in remarkably similar form in all mammals and perhaps in all vertebrates.It is hetero-dimer gene encoding glycoprotein which presents peptides to T4 cell, and plays a central role in immune system.This research studied the cloning, expression, polymorphism and the associations between some genotypes and resistance ability of major histocompatibility complex(MHC)using the methods of PCR, RT-PCR from the important freshwater fish in our country, Ctenopharyngodon idella.Our aim was to screen some MHCⅡB alleles associated with resistance in order to realize application of molecular markers in fish breeding.(1) To investigate the evolutionary patterns and population genetic variation of grass carp (Ctenopharyngodon idella) major histocompatibility complex (MHC), the DNA fragments of exon 2 and exon 3, encodingβ1 domain and P2 domain of MHC ClassⅡP respectively, and the intron between them from three grass carp populations of collected from Yangtze River were cloned and sequenced. A total of 34 alleles was defined from 572 clones, each including an exon 2 of 130-136bp, an exon3 of 218bp and an intron of 81-371bp in length. Analysis of alignments of nucleotide acid and putative amino acid sequences revealed that there were 106 variable nucleotide acid (78%) and 40 amino acid (88%) sites in exon 2,100 variable nucleotide acid (45%) and 41 amino acid (56%) sites in exon 3, suggesting more variability inβ1 than inβ2. Two phylogenetic trees constructed separately from sequences ofβ1 andβ2 domains showed 5 distinct lineages with high bootstrap values, considering the different introns of alleles in lineages, which indicated it should harbor 5 loci at least in grass carp. The numbers of synonymous substitutions per synonymous site (dS) and of nonsynonymous substitutions per nonsynonymous site (dN) were calculated for peptide binding residues (PBR) and non-PBR of theβ1 domain andβ2 domain. The results showed the ratio of dN to dS in PBR was 2.03 (P<0.05), whereas ratios in non-PBR andβ2 domain were lower than 1, implying divergent selection impacted at MHC of grass carp. Analysis of molecular variance (AMOVA) based on allele frequency, FST=0.37%, revealed little genetic differentiation among populations of grass carp in Yangtze River. (2) Semiquantitative RT-PCR demonstrated that high expression was detected in kidney, intestine and heart, low expression in muscle and the brain was not expressed. Challenge of Grass Carp with pathogenic bacteria, Aeromonas hydrophila,resulted in a significant decrease and rise in the expression of MHC classⅡB mRNA from 5 h to 88h after infection in liver,kidney,heart,intestine and gill except spleen in which the expression maintained the upward trend,followed by a recovery to normal level after 112h,and the expression still kept up a high level in intestine at 112h.
        

长江草鱼群体MHC Class Ⅱ B基因的克隆、表达及多态性分析

摘要6-8
ABSTRACT8-9
第1章 文献综述10-24
    1 MHC的发现10
    2 MHC的结构和功能10-12
    3 MHC分子进化特点12-16
        3.1 高度多态性12-14
        3.2 连锁不平衡14-15
        3.3 基因倍增与重组15-16
    4 MHC在渔业上的应用16-22
        4.1 脊椎动物适应性免疫系统的分子进化16-19
        4.2 种群及生态遗传学19-21
        4.3 抗病与遗传育种21-22
    5 本研究的目的、意义和研究思路22-24
第2章 长江草鱼群体MHC Class Ⅱ B基因的表达、多态性和进化分析24-51
    1 材料与方法25-36
        1.1 材料25
            1.1.1 多态性与进化分析材料25
            1.1.2 表达材料25
        1.2 实验仪器、试剂及溶液的配制25-28
            1.2.1 实验仪器25-26
            1.2.2 实验试剂26
            1.2.3 溶液配制26-28
        1.3 总DNA的提取28
        1.4 草鱼总RNA的提取28-29
        1.5 总RNA的消化(RQ1 RNase-Free DNase,Promega)29
        1.6 第一链cDNA的合成(M-MLV Reverse Transcriptase,Promega)29-30
        1.7 半定量RT-PCR30-31
            1.7.1 目的片段的RT-PCR30-31
            1.7.2 PCR产物琼脂糖凝胶电泳检测31
        1.8 目的基因的克隆31-35
            1.8.1 目的片段的扩增31-32
            1.8.2 目的片段的回收纯化(Takara)32-33
            1.8.3 连接反应(Takara)33
            1.8.4 感受态细胞的制备与转化(CaCl2法)33-34
            1.8.5 克隆的筛选34-35
        1.9 数据分析35-36
    2 结果与分析36-46
        2.1 序列的多态性36-37
        2.2 选择压力检测37-38
        2.3 等位基因的系统关系38-42
        2.4 种群遗传结构42
        2.5 用RT-PCR检测草鱼MHC Ⅱ B基因的表达42-46
            2.5.1 RT-PCR检测草鱼MHC Ⅱ B基因在正常组织中的表达42-44
            2.5.2 RT-PCR检测草鱼MHC Ⅱ B基因在病原菌感染后组织中的表达44-46
    3 讨论46-51
        3.1 草鱼MHC ClassⅡ B基因的进化模式47-48
        3.2 种群变异及应用意义48-49
        3.3 草鱼MHC Ⅱ B的表达分析49-51
参考文献51-63
硕士期间完成的论文63-64
致谢64
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