扬子鳄T细胞受体基因的基因组结构与进化分析

发布时间：2018-06-23 09:09

  本文选题：扬子鳄 + TCR　； 参考：《中国农业大学》2017年博士论文

【摘要】：T细胞在动物适应性免疫系统中发挥重要的作用,主要参与细胞免疫,具有信号传递、激活B细胞、杀伤靶细胞、调节免疫应答等功能,T细胞特异性免疫功能的行使需要通过T细胞表面受体(T cell receptor,TCR)来完成。之前的研究发现有颂类脊椎动物中都存在α、β、γ和δ四种典型的TCR基因类型,其蛋白产物可以形成αβ和γδ两种跨膜型异源二聚体。此外,在低等哺乳动物、鸟类、两栖类和软骨鱼中还存在非典型的TCR基因类型:TCRμ、VHδ(位于TCRα/δ位点的与免疫球蛋白重链可变区序列相似性更高的V基因)和NAR-TCR。爬行类动物在进化过程中是连接两栖类、鸟类和哺乳动物的纽带,目前对其TCR基因的研究还非常少,在很大程度上限制了我们对有颌类脊椎动物TCR基因进化模式和过程的理解。鳄目动物是现存较为原始的爬行类动物,同时被广泛认为具有强大的免疫系统。本研究选择中国特有的扬子鳄(Alligator sinensis)为研究对象,对其TCR基因进行了全面的分析。本研究首先利用鸟类TCR基因恒定区氨基酸序列在已经公布的扬子鳄基因组序列中进行搜索,得到扬子鳄的TCR基因恒定区的序列信息;再通过筛选扬子鳄基因组BAC(bacterial artificial chromosome)文库,对得到的含有TCR基因的17条BAC克隆中的9条进行全长测序并与基因组序列进行拼接,获得了包含扬子鳄TCRβ基因全长(约500Kb)、TCRγ基因全长(109Kb)、TCRα/δ基因大部分V区和全部C区(包含两个位点,位点A跨度约1 Mb,位点B长约400 Kb)的序列信息。结合RACE(rapid amplification of cDNA ends)实验获得的转录本序列信息和Southern blotting实验结果对获得的基因组序列进行分析并绘制基因位点图谱,发现扬子鳄TCRβ基因座的结构为 Vβ(39)-Jβ1-ψCβ1-Dβ2-Jβ2(11)-Cβ2-Vβ(4),TCRγ 基因座的结构为 Vγ(18)-Jγ(9)-Cγ。与其他物种相似,扬子鳄的TCRδ基因也位于TCRα基因座内,共有两个结构相似的TCRα/δ基因位点。位点A的结构为 V(104)-Dδ1(3)-Jδ1(3)-Cδ1-Jα1(91)-Cα1-VHδ(2)(包含 80 个 Vα/δ 和 26 个 VHδ),位点 B 的结构为 V(39)-Dδ2(3)-Jδ2(3)-Cδ2-Vδ-VHδ-Jδ3-ψCδ3-VHδ(3)-Dδ4-Jδ4-Cδ4-VHδ(2)-Dδ5-Jδ5-Cδ5-Jα2(63)-Cα2(包含37个Vα/δ和11个VHδ)。RACE实验证明位点A和位点B是相邻的,因为两个位点可以共用部分可变区基因,但基因组序列搜索和BAC克隆筛选暂时都无法确定其位置关系。表达水平的分析发现扬子鳄的所有TCR基因在表达过程中都不发生体细胞超突变(somatic hypermutation,SHM),但其CDR3中存在大量的N/P核苷酸。δ链能够使用至少50%的Vα基因和大部分有功能的VHδ基因,还能同时使用多个D基因,使其CDR3的序列和长度多样性大大增加。定量PCR实验表明在外周淋巴组织中γδTCR的表达量与αβ TCR的表达量基本相同,在小肠上皮等粘膜组织中γδ TCR的表达量更高。扬子鳄的TCRβ位点和TCRγ位点与其他物种在相应的基因位点上存在同线性关系,位点结构和表达机制与其他物种基本相同。扬子鳄的TCRα/δ位点中存在大量与Cδ共同表达的VHδ基因,这些VHδ基因主要分为三组:第一组(VHδⅠ)与鸟类中的VHδ相似性较高(VHClanⅠ);第二组(VHδⅡ)与鸭嘴兽和负鼠中的VHδ和Vμ相似性较高(VH Clan Ⅲ);第三组(VHδⅢ)与鳄目动物自身的IGHV(免疫球蛋白重链可变区基因)相似性最高(VH Clan Ⅰ、Ⅱ、Ⅲ中都存在),但多数为假基因,没有发现与Cδ基因共同表达的转录本。综合序列比对、进化分析和同线性分析的结果,本研究提出了羊膜动物TCRα/δ位点的一种可能的进化过程:爬行类、鸟类和哺乳类的共同祖先中只存在一个TCRα/δ位点,并且含有VHδ Ⅰ和VHδⅡ基因(VHδ Ⅰ也可能出现在哺乳类与爬行类分化之后),在低等哺乳类中保留了部分VHδ Ⅱ,并以此分化出TCRμ基因,在高等哺乳类中丢失了全部VHδ;爬行类与鸟类的共同祖先中发生了Cδ基因的复制(鳄目动物的Cδ基因分为三组:CδⅠ、CδⅡ和CδⅢ),鸟类中保留了部分VHδⅠ和CδⅠ,并复制出一个新的VHδ-Cδ位点;鳄目动物保留了 VHδ Ⅰ和VHδ Ⅱ,还出现了 VHδⅢ,并且发生了一次TCRα/δ位点的整体复制。扬子鳄的TCRα/δ位点B更加保守和古老,与其他物种的TCRα/δ位点存在同线性关系,位点A是鳄目动物出现后由位点B复制形成的,只保留了 CδⅠ,但TCRα/δ位点A的基因在淋巴组织中的表达量远高于位点B的基因,且位点A的V、J基因数量更多,在表达水平上的使用频率也更高。综上所述,本论文对扬子鳄TCR基因的位点结构、表达机制和进化特点等进行了多方面的研究,并总结了羊膜动物TCRα/δ位点的进化模式,这些研究结果为探索VHδ基因的起源和TCR基因的进化过程提供了重要的线索。
[Abstract]:T cells play an important role in the adaptive immune system of animals. They are mainly involved in cellular immunity, signaling, activating B cells, killing target cells, regulating immune response and so on. The specific immune function of T cells is performed through the T cell surface receptor (T cell receptor, TCR). There are four typical TCR genotypes of alpha, beta, gamma and delta in animals, and their protein products can form two transmembrane heterogenous two polymers of alpha and gamma. In addition, there are atypical TCR gene types in lower mammals, birds, amphibians, and cartilaginous fish: TCR mu, VH Delta (at the TCR alpha / delta loci and the variable sequence of immunoglobulin heavy chains) The higher V gene and the NAR-TCR. reptiles are linked to amphibians, birds and mammals in the evolution process, and the study of their TCR genes is still very few, to a great extent, it restricts our understanding of the evolution patterns and processes of the TCR gene in the vertebrate vertebrates. Reptiles are widely considered to have strong immune systems. This study selected the Chinese alligator (Alligator sinensis) as the research object and analyzed its TCR gene in a comprehensive way. First, the study was conducted by using the sequence of the constant region amino acid of the bird TCR gene in the published genome sequence of the alligator. The sequence information of the TCR gene constant region of the alligator was obtained. By screening the BAC (bacterial artificial chromosome) library of the alligator genome, 9 of the 17 BAC clones containing the TCR gene were fully sequenced and spliced with the genome sequence to obtain the full length (about 500Kb), TCR gamma, containing the TCR beta gene of the alligator Alligator. The whole length (109Kb), the TCR alpha / delta gene most V and all C regions (including two loci, the site A span of about 1 Mb, the site B length about 400 Kb), and the transcriptional sequence information obtained from the RACE (rapid amplification of) experiment and the results of the genomic sequence were analyzed and plotted. The structure of the TCR beta loci of the Chinese alligator was found to be V beta (39) -J beta 1-, C beta 1-D beta 2-J beta 2 (11) -C beta 2-V beta (4), TCR gamma loci were V gamma (18) -J gamma (9). Adadjacent adadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadbecause of the position Some variable region genes can be shared, but genomic sequence search and BAC clone screening can not determine its position relationship temporarily. Analysis of expression level shows that all TCR genes in alligator Alligator do not have somatic hyper mutation (somatic hypermutation, SHM) during the expression process, but there are a large number of N/P nucleotides in CDR3. Delta chains can be found in CDR3. The use of at least 50% of the V alpha gene and most of the functional VH delta genes can also use multiple D genes at the same time, making the sequence and length diversity of the CDR3 greatly increased. Quantitative PCR experiments show that the expression of delta TCR in peripheral lymphoid tissues is basically the same as the expression of alpha beta TCR, and the expression of delta TCR in small intestinal epithelium and other mucosa tissues. The TCR and TCR gamma loci of the alligator and other species have the same linear relationship with other species, and the structure and expression mechanism of the loci are basically the same as those of other species. There are a large number of VH delta genes in the TCR alpha / delta loci of alligator Alligator, which are mainly divided into three groups: the first group (VH delta I) and the birds. The VH delta similarity is higher (VHClan I); the second group (VH Delta II) is similar to the VH Delta and V in the duckbill and the opossum (VH Clan III), and the third group (VH delta III) is the highest (VH Clan, II, and III) of the crocodile itself (VH Clan, II, and III), but most of them are pseudogenes and are not found with the C delta base. A possible evolutionary process of the TCR alpha / delta loci of amniotic amniotic animals was proposed by the co expression of transcriptional transcripts, integrated sequence alignment, evolutionary analysis and linear analysis. The common ancestor of reptiles, birds and mammals had only one TCR alpha / delta locus, and the VH delta I and VH Delta II genes (VH delta I may also appear. After the differentiation of mammals and reptiles), some VH Delta II was retained in the lower mammals, and the TCR gene was differentiated and all VH delta was lost in the higher mammals; the C delta gene was replicated in the common ancestor of reptiles and birds (the C delta gene of the crocodile was divided into three groups: C delta I, C Delta II and C delta), which were retained in birds. Part VH delta I and C delta I, and replicate a new VH Delta -C delta loci; the crocodile retained the VH delta I and VH Delta II, and also appeared VH delta III, and a TCR A / delta locus was produced as a whole. The TCR alpha / delta loci of the alligator are more conservative and old, and have the same linear relationship with the TCR alpha / delta loci of other species, and the loci A is a crocodile movement. Only C delta I was retained after the occurrence of site B, but the expression of TCR alpha / delta locus A in lymphoid tissues is much higher than that of the loci B gene, and the V, J gene number of A and the frequency of use at the expression level are higher. In this paper, the structure, expression mechanism and evolution of the TCR gene in alligator Alligator are described in this paper. Many studies have been carried out and the evolution patterns of TCR alpha / delta loci of amniotic animals have been summarized. These results provide important clues for exploring the origin of VH delta gene and the evolution of TCR genes.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q953
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本文编号：2056617