文昌鱼REL、NFAT、TGFBI基因克隆表达及TLR信号通路进化研究

【作者】 宋晓军；

【导师】 马飞；

【作者基本信息】 南京师范大学 ， 动物学， 2014， 博士

【摘要】 文昌鱼作为脊椎动物的祖先物种,对研究脊椎动物形态发生、发育和免疫系统的进化过程起到关键作用。随着大量物种基因组测序的完成与比较免疫学的迅速发展,以及佛罗里达文昌鱼全基因组测序的完成,为我们系统的研究脊椎动物免疫系统的起源与进化提供了物质基础。因此,本论文以文昌鱼EST数据库(http://rich.yunda.org/test/amphioxusest/)和一些公共数据库为基础,对白氏文昌鱼(Branchostoma belcheri)REL、NFAT、GFBI基因的功能与进化进行研究；并利用比较基因组学的研究方法对动物TLR信号通路的起源与进化进行了详细的研究,从而揭示自然选择在基因复制、基因结构的改变和结构域变化过程中的作用。具体工作如下：1、哺乳动物核因子活化B细胞K轻链增强子(nuclear factor kappa-light-chain-enhancer of activated B cells,NF-κB)蛋白家族基因是一类非常重要的转录因子,在信号传递、细胞凋亡、先天性免疫和适应性免疫等过程中起到关键的作用。我们在白氏文昌鱼基因组中克隆了一个NF-κB蛋白家族基因(命名为AmphiREL),该基因全长2700bp,编码773个氨基酸,含有RHD和IPT两个结构域。在LPS免疫刺激下能够参与到文昌鱼的先天性免疫反应过程中,在刺激后6h时表达量达到最高,为对照组(注射PBS)的2.4倍。基因共线性与基因结构比较分析结果显示文昌鱼AmphiREL基因与人类的RELB基因直系同源,与人类的RELA和REL基因是旁系同源关系。在进化过程中Rel亚家族基因的保守motif发生规律性的改变,并受到较强的纯化选择。我们的研究结果为更好的理解Rel亚家族基因的功能和进化提供了重要线索。2、活化T细胞核因子(Nuclear Factor of Activated T cells,NFAT)最早是在细胞核提取物中被发现,它是一类能够结合在白细胞介素-2(IL-2)上的可诱导的DNA结合因子。NFAT在免疫系统和调节脊椎动物发育过程中起到非常重要的作用。我们首先在白氏文昌鱼基因组中克隆了一个NFAT蛋白家族基因(命名为AmphiNFAT),其基因全长为3547bp,编码605个氨基酸,包含一个非常保守的DNA结合结构域(RHR domain)、一个中度保守的调节结构域(NHRdomain)、和一个不太保守的C端结构域。在LPS免疫刺激下,该基因能够参与文昌鱼的先天性免疫反应过程。空间表达模式分析结果显示,文昌鱼AmphiNFAT基因在肌肉和脊索中表达量最高,在鳃中表达量次之,在肠、肝盲囊和性腺中表达量最低。进化研究结果表明NFAT蛋白家族基因最早存在于腔肠动物门动物的共同祖先中,并且在进化过程中文昌鱼和海胆基因组丢失了NFAT1-4基因。另外,在进化过程中NFAT蛋白家族基因受到较强的纯化选择。我们提出NFAT蛋白家族基因的复制及新功能的产生与自然选择压力的关系模型。我们的结果为更好的理解NFAT蛋白家族基因的功能和进化提供有力的证据。3、转化生长因子β诱导基因(transforming growth factor β induced gene,TGFBI)最初是在TGFβ1诱导的肺癌细胞系中被发现,它能够编码一个细胞外基质(ECM)蛋白。我们首先在文昌鱼基因组中克隆了一个TGFBI蛋白家族基因(命名为AmphiTGFBI),该基因全长3095bp,编码867个氨基酸,包含一个信号肽序列、一个EMI结构域和5个Fas1结构域,然而其他物种中最多含有4个Fas1结构域。组织表达模式分析结果显示,文昌鱼AmphiTGFBI基因在所有检测的5个组织中都有表达。进化研究结果表明TGFBI蛋白家族成员最早出现在海绵动物的共同祖先基因组中,并且无脊椎动物TGFBI蛋白家族成员的基因结构和保守motif都存在规律性的变化。这些变化可能是由TGFBI蛋白家族成员在进化过程中Fas1结构域的丢失和一些分枝受到正选择的作用导致的。这些研究结果为深入研究TGFBI蛋白家族成员的功能与进化提供了理论依据。4、Toll样受体信号通路(Toll-like receptor signaling pathway)的网络结构和功能从昆虫到哺乳动物都是非常保守的,并且在果蝇和哺乳动物的先天性免疫反应过程中起到关键作用。另外,基因在行使其生物学功能过程中往往需要大量蛋白分子相互作用的复杂网络来实现的。因此,我们在网络水平上对TLR(Toll-like receptor)信号通路的起源与进化进行了研究。其研究结果表明TLR信号通路相关基因在进化过程中受到较强的纯化选择压力,并且这些基因受到的选择压力跟基因在通路中所处的位置成反比。另外,TLR信号通路可能最早出现在海绵和早期多细胞动物共同祖先的基因组中,并在进化过程中伴随着TLR、IKK、IκB、NF-κB等基因的复制和接头分子的扩增,以及NF-κB基因的结构和保守结构域的变化。这些结果能够更好的帮助我们理解动物TLR信号通路的进化历史和网络结构之间的联系。本论文对文昌鱼基因REL,NFAT,TGFBI的功能和进化及TLR信号通路的进化和起源问题进行了研究,率先揭示了REL、NFAT、TGFBI和TLR信号通路的起源和进化过程。对自然选择在基因复制,基因结构的改变及保守结构域的变化过程中所起的作用进行了探讨,为我们更好地理解自然选择与基因新功能的获得奠定了基础。更多还原

【Abstract】 Amphoxus, as an ancestral species of vertebrate animals, play important roles in the evolution of vertebrate morphogenesis, development, immunity. With the development of whole genome sequencing finished, the rapid development of the comparative immunology and the completion of Branchostoma florida genome sequencing which could provide the material basis for systematically studying the evolution and the origin of vertebrate immune system. Therefore, we studied the function and evolution of amphoxus(Branchostoma belcheri) REL, NEAT, TGFBI genes based on the amphioxus EST database (http://rich.yunda.org/test/amphioxusest/) and some public database and then we used the comparative genomics research methods to study the evolution and origin of TLR signaling pathway in order to reveal the relationship between the natural selection and gene duplication, the shifted of gene structure, and the changed of gene domains. The detail results as following:1. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), as a very important transcription factor, play an important roles in cell signalling, apoptosis, innate immunity and adaptive immunity and so on. We cloned a NF-κB gene from Branchostoma belcheri genome (named as AmphiREL). Its full-length has2700bp and encodes a putative protein with773aa, which including two conserved domains:RHD domain and IPT domain. AmphiREL could involve in amphioxus innate immunity after LPS stimulation, which gene expression was significantly up-regulated up to2.4fold (P<0.01) at6h after LPS challenge. The results of gene synteny and gene structure analysis have shown that AmphiREL gene may be orthologous to human RELB, whereas human RELA and REL are paralogous genes of the AmphiREL gene. The conserved motif of Rel subfamily genes has regular change and all genes under very strong negtive selection in the evolutionary history. Together, our results provide important clues for understanding the evolution and function of Rel subfamily genes.2. NFAT (Nuclear Factor of Activated T cells), which paly an important roles in immunity system and development of vertebrate, was first identified in nuclear extracts of activated T cells as an inducible DNA-binding factor that binds to IL-2. We cloned a NFAT gene from Branchostoma belcheri genome (named as AmphiNFAT) which full-length has3547bp, encodes a putative protein with605aa. The polypeptide has a highly conserved DNA-binding domain (Rel homology region, RHR), a moderately conserved regulatory domain (NFAT homology region, NHR), and a non-conserved carboxyterminal domain. The AmphiNFAT could response to LPS-induced innate immunity of amphioxus. Tissue expression pattern of AmphiNFAT gene has shown that the expression of AmphiNFAT was high in the muscles and notochord, moderate in gonad and gills, and low in the intestine and hepatic cecum. The NFAT family genes were present in a common ancestor with cnidaria, and NFAT1-4paralogs were lost early in Branchiostoma and Strongylocentrotus genomes. Besides, we discovered that NFAT family genes underwent strong purifying selection. Finally, we propose a model of the relationship between natural selection and NFAT genes duplication. Taken together, our findings provide an insight into the innate immune response of amphioxus and the evolution of the NFAT gene family.3. TGFBI (transforming growth factor β induced) encoding an extracellular matrix (ECM) protein, was identified from a TGFβ1treated lung adenocarcinoma cell line. We cloned a TGFBI gene from Branchostoma belcheri genome (named as AmphiTGFBT), which full-length has3095bp, encodes a putative protein with867aa. The AmphiTGFBI contains a signal peptide (SP), an EMI domain, five Fasl domains (Fasla-e) compared to no more than4Fasl domains in other animals. The tissue expression patterm has shown that AmphiTGFBI was detected in all tissues. The TGFBI family genes were present in a common ancestor with Amphimedon queenslandica and the gene structures and conserved motifs of invertebrate TGFBIs were found to present regular changes during the evolutionary history. Positive selection and Fasl domain loss might cause the regular changes of gene structures and conserved motifs in invertebrate TGFBI during the evolutionary history. Together, our findings provided an insight into the function and evolution of the TGFBI family.4. The TLR signaling pathway, which plays a central role in innate immunity from Drosophila to mammal, is highly conserved in structure and function from insects to vertebrates. Otherwise, genes carry out their biological functions through pathways in complex networks consisting of many interacting molecules. Therefore, we studied the evolution and origin of animal TLR signaling pathway revealed by network-level molecular evolutionary analyses. All genes in TLR signaling pathway were highly conserved and underwent strong purifying selection and the selection constraint of genes was negatively correlated with its position along TLR signaling pathway. The TLR signaling pathway might present in a common ancestor of sponges and eumetazoa, and evolve via the TLR, IKK, IκB and NF-κB genes underwent duplication events as well as adaptor molecular enlargement, and gene structure and conservation motif of NF-κB genes shifted in their evolutionary history. Our results will improve our understanding on the evolutionary history of animal TLR signaling pathway as well as the relationship between the network architecture and the sequences evolution of individual protein.In this thesis, the function and evolutionary history of amphioxus REL, NFAT, TGFBI genes and the evolution and origin of animal TLR signaling pathway have been studied. We first revealed the proccess of evolution and origin of Rel subfamily, NFAT family, TGFBI family and TLR signaling pathway, and discussed the relationship between natural selection and gene duplication, the change of gene structure and the change of conserved domain. Our results will improve our understanding on the relationship between natural selection and new function obtained of proteins.更多还原