长牡蛎甲状腺激素及其信号通路关键基因的功能研究

IOCAS-IR > 海洋生物技术研发中心

	长牡蛎甲状腺激素及其信号通路关键基因的功能研究
	黄雯1,2
学位类型	博士
导师	张国范 ; 阙华勇
	2015-05-23
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	水产养殖
关键词	长牡蛎甲状腺激素核受体
摘要	长牡蛎（Crassostrea gigas），生活于潮间带，是全世界重要的水产养殖种类之一。由于其特殊的生态位和较高的经济价值，长牡蛎已成为冠轮动物中得以研究最多的物种之一。在长牡蛎中研究甲状腺激素及其信号通路关键基因不仅有助于加深了解冠轮动物，也为解析无脊椎动物和脊椎动物的内分泌系统进化过程、促进牡蛎产业技术进步提供宝贵的科学信息。本论文的研究内容包括三大块，主要内容和结论如下： 1长牡蛎甲状腺激素及其信号通路关键基因的功能研究上个世纪，甲状腺激素（THs，包含T4和T3）被认为只在脊椎动物中发挥作用。近些年，越来越多的证据表明，THs也存在于头索和尾索动物中。然而，目前THs相关的研究在非脊索的无脊椎动物还很少见。为了研究THs的起源与进化，我们选择了冠轮动物的代表—牡蛎为研究对象，通过生理和分子两种手段探讨牡蛎中的THs系统。本研究用高效液相色谱法和液相色谱与质谱串联的方法定性的检测了T4和T3的存在，又用电化学免疫法定量检测了牡蛎发育过程中THs的变化。使用分子手段克隆了一些THs信号通路中的关键基因，包括4个合成酶甲状腺过氧化物酶（PERT），2个代谢酶脱碘酶，一个甲状腺激素受体（TR）。使用荧光定量分析、免疫印迹分析、亚细胞定位、原核表达、酵母双杂交、凝胶迁移电泳(EMSA)、双报告基因技术等一系列分子生物学实验技术，对这些基因进行了功能鉴定。实验数据证明，在牡蛎中也存在甲状腺激素及其信号通路。THs可能参与胚胎形成，生长和变态等一些发育过程。牡蛎胚胎可能通过CgPERT1自体合成THs。牡蛎胚胎可以在体内将T4转化为T3，这项功能可能是由脱碘酶基因（CgDx或CgDy）行使的。CgDx和CgDy的mRNA表达受到THs和CgTR的调控，它们的启动子区域含有TRE。THs可能通过CgTR与TRE结合来调控CgDx和CgDy的转录。这些实验证据说明TH系统的反馈调节机制在牡蛎中也是存在的。 CgTR能通过与目的基因的启动子区域结合来调控基因的表达，并能与视黄酸X受体（CgRXR）发生相互作用。CgTR mRNA的表达受到T4和CgTR蛋白的调控，且在CgTR的启动子区域找到了一个甲状腺激素效应元件（TRE）。这些CgTR的功能与脊椎动物的TR一致，说明这些功能在TR的共同祖先中已经存在。然而，CgTR的DNA结合特性和转录激活活性并不保守。与血吸虫的TR相似，CgTR能与DR0-DR5结合。此外，两种双报告实验证明CgTR的转录激活活性不能被T4, T3或TRIAC激活。这些不保守的功能将为TR功能乃至TH系统的进化提供宝贵的线索。这是首次在无脊椎动物中系统地研究THs的生理作用和分子机理。根据本研究获得的结果，我们认为THs及其信号通路在牡蛎中是存在的，可将THs的起源追溯至冠轮动物。 2视黄酸X受体RXR的功能研究本研究克隆获得了牡蛎中唯一的RXR基因CgRXR。该基因在胚胎发育过程中表达量较高，说明可能参与到牡蛎的胚胎发育过程。该基因定位于细胞核，为其作为转录因子调控基因表达提供空间便利。通过荧光双报告实验证明CgRXR的转录激活活性可以被9-cis RA、DHA激活，说明CgRXR LBD的结合功能是保守的。CgRXR的转录激活活性也可以被环境污染物三丁基锡（TBT）、三苯基锡（TPT）激活，说明有机锡污染物对牡蛎的毒害作用可能是通过RXR来介导的。EMSA实验结果表明，CgRXR能与DR0-DR5结合，除了与DR4结合较弱外，与其余的探针结合能力相当。双壳类RXR的DNA结合特性不保守，与脊椎动物和腹足类的均有较大差异。 3新的核受体亚家族NR8的进化分析和CgNR8A1的功能研究核受体（NR）属于转录因子超级族，它通过调节基因表达来调控后生动物的发育、内稳态、分化和繁殖等。近几年，迅速发展的基因组计划为核受体的进化和功能研究提供了新的机遇。具有典型结构的核受体被分为6个亚家族。本研究在长牡蛎中克隆了一个具有典型结构的核受体（CgNR8A1）。然而进化分析发现该基因不能归类于现有的NR亚家族。通过数据挖掘，我们在后生动物中（包括刺胞动物，软体动物，环节动物，棘皮动物，半索动物和头索动物）又找到了9个CgNR8A1的同源基因。进化分析发现它们组成了一个新的NR亚家族，命名为NR8亚家族。NR8是第三出现的NR亚家族，起源于真后生动物的共同祖先，并在脊椎动物、蜕皮动物、扁形动物的祖先中分别独立发生了基因丢失事件。对CgNR8A1的功能研究结果表明，CgNR8A1具有核定位信号肽GKHRNKKPRLD，并定位于细胞核。发育过程中的表达模式暗示CgNR8A1可能参与胚胎的形成。EMSA实验显示CgNR8A1能与保守的DNA核心基序DR0、DR2、DR4紧密结合，与DR1、DR3、DR5、Half和Pal0结合较弱。新鉴定的NR8亚家族增加了人们对NR亚家族进化的理解，CgNR8A1的功能鉴定有助于对NR8亚家族功能的理解。
其他摘要	The Pacific oyster (Crassostrea gigas), living in the intertidal area, is a world-wide aquaculture species. Due to its remarkable ecological and economic value, the Pacific oyster has become one of the most well-known species in lophotrochozoa. In this study, we evolutionary studied the Thyroid hormone system and nuclear receptor family members in the Pacific oyster. The following were the main results and conclusions: 1 The functional study of thyroid hormone and critical genes of its singnaling pathway in the Pacific oyster Crassostrea gigas Thyroid hormones (THs) play important roles in development, metamorphosis, and metabolism in vertebrates. During the past century, TH functions were regarded as a synapomorphy of vertebrates. More recently, accumulating evidences have gradually convinced us that TH functions also occur in invertebrate chordates. To date, however, the TH-related study in non-chordate invertebrates is limited. In this study, THs were qualitatively detected by two reliable methods (HPLC and LC/MS) and quantitative measured by a electrochemical immuno-methods in the development of C. gigas. Molecular biology methods were used to study the critical genes in the THs signaling pathway, including four thyroid hormone peroxidases (PERT), two iodothyronine deiodinase and one throid hormone receptor (TR). The gene function of them were characterized by various molecular biology methods, including qRT-PCR, western blot, subcellular localization, prokaryotic recombinant protein expression, yeast two-hybrid assay, electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay. The results suggested that there is an active TH system in oysters. THs may involve in embryogenesis, growth and metamorphosis of C. gigas. Oyster embryos may synthesize THs autonomously via CgPERT1. The deiodinase activity of transformation from T4 to T3 was detected in the Pacific oyster C. gigas in vivo and the identified oyster deiodinases (CgDx and CgDy) were speculated to be responsible for this deiodinase activity. The promoters of CgDx and CgDy were activated by CgTR in a dual-luciferase reporter system. Furthermore, the atypical TH responsive elements (TREs) in the promoters could be bound by recombinant CgTR. Thus, CgDx and CgDy are also TH-responsive genes and THs may transcriptional regulate the expression of oyster deiodinases via CgTR binding to TREs in their promoters, suggesting a conserved TH feedback regulation mechanism in mollusks for the first time. As a first step in elucidating the TH signaling cascade, an ortholog of vertebrate TH receptor (TR), the most critical gene mediating TH effects, was cloned in C. gigas. The sequence of CgTR has conserved DNA-binding and ligand-binding domains that normally characterize these receptors. Experimental results demonstrated that CgTR can regulate gene expression through binding to promoters of target genes and can interact with oyster retinoid X receptor (CgRXR). Moreover, CgTR mRNA expression was regulated by TH and CgTR proteins, and an atypical thyroid hormone response element (CgDR5) was found in the promoter of CgTR and verified by EMSA assay. These results indicated that some of the CgTR function is conserved, and suggest that these TR functions had developed in the common ancestor of protostome and deuterostome TR. However, differences were also found in the DNA binding specificity and transcriptional activation activity of CgTR when compared with vertebrate TRs. the functions of CgTR provide significant evidence in understanding evolution of the TH system. This is the first systematic study on physiological effects and molecular mechanism of THs in non-chordate invertebrates. On the basis of our findings in this study and the sporadic evidence from other lophotrochozoa, it is reasonable to conclude that the origin of THs could date back to the lophotrochozoa. 2 Functional characterization of CgRXR In this study, we also cloned another NR member, CgRXR. qRT-PCR results indicated that CgRXR may be involved in embryogenesis in C. gigas. A recombinant full-length protein of CgRXR localized to the nuclei of HeLa cells. Dual-luciferase reporter assay results indicated that 9-cis RA or DHA could activate the transcriptional activation activity of CgRXR. This means that the ligand binding activity of CgRXR is conserved in C. gigas. Furthermore, TBT or TPT could activate the transcriptional activation activity of CgRXR, suggesting that the toxicity of organotin compound to oysters were madiated by CgRXR. CgRXR could binding to DR0-DR5, except for the weakness binding of DR4, the binding intensity of the other probes were comparable. Thus, the DNA binding property of CgRXR is different from vertebrates and gastropods. 3 Evolution of a novel nuclear receptor subfamily with emphasis on the member from the Pacific oyster Crassostrea gigas Nuclear receptors (NRs) belong to the transcription factors superfamily that regulates development, homeostasis, differentiation, and reproduction in metazoans via control of gene expression. Recently, rapid advances in genome projects for various metazoans provided new opportunities to study the evolution and function of NRs. Typically structured NRs are divided into six subfamilies. Here, the gene for a typically structured NR (CgNR8A1) was cloned from the Pacific oyster Crassostrea gigas. However, this novel receptor could not be assigned to one of the known NR subfamilies. Through data mining, nine other CgNR8A1 gene homologs were identified in metazoans, including cnidarians, mollusks, annelids, echinoderm, hemichordate, and cephalochordate. Phylogenetic analysis showed that these receptors were part of a novel NR subfamily, herein designated as NR8. Evolutionary analysis revealed that the NR8 subfamily was phylogenetically the third-oldest NR subfamily, originating from a common ancestor of Eumetazoa, and several gene loss events occurred independently in anscestors of vetebrates, ecdysozoans and platyhelminthes, which do not have NR8 member. Furthermore, the function of CgNR8A1 was investigated to give insight into the function of this novel NR subfamily. A nuclear localization signal peptide GKHRNKKPRLD was identified in CgNR8A1 and a recombinant full-length protein of CgNR8A1 localized to the nuclei of HeLa cells. The mRNA expression profile of CgNR8A1 suggested that it may be involved in embryogenesis in C. gigas. EMSA experiments showed that CgNR8A1 bound strongly to conserved DNA core motifs DR0, DR2, and DR4 and weakly to DR1, DR3, DR5, Half, and Pal0. In summary, the novel NR8 subfamily identified here extends the evolutionary understanding of NRs, and the functional analysis of CgNR8A1 provided further insights into the function of the NR8A1s.
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/23280
专题	海洋生物技术研发中心
作者单位	1.中国科学院海洋研究所 2.中国科学院大学
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	黄雯. 长牡蛎甲状腺激素及其信号通路关键基因的功能研究[D]. 北京. 中国科学院大学,2015.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
长牡蛎甲状腺激素及其信号通路关键基因的功（5200KB）	学位论文	PDF	限制开放	ODC ODbL	浏览