生物胺类受体在长牡蛎不同发育时期中的功能研究

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	生物胺类受体在长牡蛎不同发育时期中的功能研究
其他题名	Functional Characterization of Biogenic Amine Receptors during Different Developmental Stages of the Pacific Oyster, Crassostrea gigas
	纪鹏
学位类型	博士
导师	张国范
	2017-05-20
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	海洋生物学
关键词	G蛋白偶联受体生物胺类受体长牡蛎发育时期功能研究
摘要	G蛋白偶联受体（G protein-coupled receptor，GPCR）是膜蛋白受体家族中的一大类群，也是细胞表面受体家族中成员最多的一类受体。它通过与G蛋白结合将细胞外信号传导进入细胞内部，是细胞信号转导过程中重要的媒介分子。GPCR参与了生物体内包括视觉、味觉、嗅觉、情绪的调节、行为的控制、神经系统的调节、内分泌系统的稳态、免疫系统的调节等多种重要的生理活动。因此，GPCR常被作为药物的靶标分子被广泛应用于各种制药工业中。在生物体内，大部分GPCR都属于其家族分类中的A类视紫红质家族。而在视紫红质家族中，以章鱼胺受体、酪胺受体、多巴胺受体、肾上腺素受体和血清素受体为代表的生物胺类受体参与到了包括脊椎动物和无脊椎动物在内的多种生物的重要生命活动过程中，因而获得了广泛的关注和深入的研究。生物胺类受体是一类能够与生物胺相结合的GPCR。生物胺广泛存在于动植物体内以及各种食品中，是含氮的具有生物活性的一类有机化合物。生物胺的分子量一般都比较小，但是可以参与到生物体的生长、发育、生殖等众多的重要生理活动中，是具有潜在效应的神经递质分子。作为无脊椎动物中常见的生物胺，章鱼胺（octopamine，OA）和它的前体物质酪胺（tyramine，TA）参与到了无脊椎动物的诸如生殖、性成熟、变态等众多的发育过程中，并在其中扮演着非常重要的角色。虽然OA首先在软体动物章鱼中被发现，目前针对于OA、TA以及OA/TA受体的研究工作主要集中于蜕皮动物特别是各种昆虫中。而在贝类等软体动物中，针对OA、TA以及OA/TA受体的研究则较少，它们在贝类等软体动物体内所发挥的生理功能目前也不清楚。在本文中，我们主要开展了对OA/TA受体在海洋贝类长牡蛎（Crassostrea gigas）不同发育时期中的生理功能研究。一、长牡蛎中潜在G蛋白偶联受体的家族分类及表达谱分析根据实验室之前已经测序获得的长牡蛎的全基因组序列数据，我们对已注释的长牡蛎基因组序列进行了初步筛选，共获得了553条潜在的GPCR序列。随后，通过对这些潜在的GPCR序列进行疏水性跨膜区分析和结构域筛选，最终鉴定出了412条GPCR序列。通过BLAST同源比对和系统发育分析，确定了这412条序列的家族分类情况：其中308条GPCR属于A类受体家族，44条GPCR属于B类受体家族，14条GPCR属于C类受体家族，另外还有46条GPCR序列属于未知的GPCR蛋白家族。此外，通过对长牡蛎中A类GPCR家族的序列数据进行统计和分析我们还发现，在长牡蛎的A类GPCR家族中，至少有36条GPCR序列属于生物胺类受体亚家族。随后，通过对这36个潜在生物胺类受体在长牡蛎不同发育时期的表达谱数据进行深入分析，我们将长牡蛎中大多数潜在生物胺类受体的表达模式进行了初步分类，大体分成了四种类型：（1）在长牡蛎的整个发育过程中，GPCR几乎没有表达或者表达量极低；（2）GPCR主要在长牡蛎的早期发育阶段表达；（3）GPCR主要在长牡蛎的中期发育阶段表达；（4）GPCR主要在长牡蛎的个别发育时期中特异性表达。尤其是在第4种类型中，我们还筛选鉴定出了两个能够在长牡蛎的眼点幼虫期特异性上调表达的新型生物胺类受体——CGI_10017568和CGI_10027100，这两个受体蛋白有可能参与到了长牡蛎的附着变态发育过程中。二、新型章鱼胺/酪胺受体CgGPR1在长牡蛎不同发育时期中的功能研究通过PCR扩增和RACE技术，我们从长牡蛎中克隆得到了一个潜在的OA/TA受体蛋白CgGPR1（CGI_10017568）。CgGPR1基因全长为2,826 bp，编码一个含有524个氨基酸的受体蛋白。BLAST结果显示，CgGPR1与来自椎实螺Lymnaea stagnalis的章鱼胺受体OAR2同源性最高。同源多序列比对和系统发育分析结果显示，CgGPR1属于一个研究较少的无脊椎动物新型OA/TA受体亚家族。随后，通过qPCR技术、Western Blot技术和整体免疫荧光定位技术研究了CgGPR1在长牡蛎不同发育时期和不同成体组织中的时空表达模式。结果显示，CgGPR1在长牡蛎从卵细胞期发育到稚贝的所有阶段中都有表达，在原肠胚期、担轮幼虫期和D型幼虫期的表达水平最高，这表明CgGPR1可能参与了长牡蛎的胚胎发生和组织分化过程。CgGPR1的免疫荧光信号主要出现在长牡蛎幼虫的面盘、足、鳃和外套膜处，这表明CgGPR1可能在长牡蛎幼虫的游动、滤食以及感应外界环境等生理过程中发挥着重要的作用；在长牡蛎眼点幼虫期的足表面检测到了CgGPR1的免疫荧光信号，这也表明CgGPR1可能在长牡蛎的附着变态发育过程中发挥着潜在的作用。在长牡蛎所有检测的成体组织中，均检测到CgGPR1的mRNA转录水平，在外套膜中的转录水平最高，在性腺中的转录水平最低，这暗示着CgGPR1可能在长牡蛎的成体组织中也发挥着一定的作用。此外，通过构建能够稳定过表达CgGPR1的HEK293细胞株，我们还研究了生物胺类神经递质（激动剂）对可稳定过表达CgGPR1的HEK293细胞内第二信使分子cAMP和Ca²⁺水平的影响。结果显示，OA、TA以及其他相关的生物胺类物质不能引起可表达CgGPR1的HEK293细胞内cAMP和Ca²⁺的浓度发生变化，这说明CgGPR1及其所代表的第四亚家族无脊椎动物OA/TA受体蛋白可能并不参与cAMP和Ca²⁺所介导的细胞信号转导通路，而是参与了其他未知的信号分子所介导的转导通路。我们对CgGPR1的研究为后续进一步地开展对CgGPR1及其近源蛋白在无脊椎动物中的生理功能的研究奠定了良好的基础，也为后续人工干预或调控海洋贝类等污损生物的附着变态过程提供了潜在的靶标分子。三、新型酪胺受体CgTAR1在长牡蛎不同发育时期中的时空表达模式分析我们还从长牡蛎中克隆了一个潜在的TA受体蛋白CgTAR1（CGI_10027100）。CgTAR1基因全长为1,601 bp，编码一个含有407个氨基酸的受体蛋白。BLAST结果显示，CgTAR1与来自蝗虫Locusta migratoria的酪胺受体TAR1同源性最高。同源多序列比对分析和系统发育分析结果显示，CgTAR1属于无脊椎动物OA/TA受体家族中类α₂-肾上腺素的酪胺受体亚家族。随后，通过qPCR技术、Western Blot技术以及整体免疫荧光定位技术研究了CgTAR1在长牡蛎不同发育时期和不同成体组织中的时空表达模式。结果显示，CgTAR1 mRNA在长牡蛎桑椹胚期、囊胚期和眼点幼虫期有较高的转录表达水平，而在D型幼虫期和壳顶幼虫期几乎没有或者只有少量的表达。在蛋白水平上，CgTAR1在长牡蛎从卵细胞发育到D型幼虫期中均有大量的表达，表明CgTAR1可能参与长牡蛎的胚胎发生、组织分化和器官形成过程，在长牡蛎的早中期发育过程中起着重要的作用。CgTAR1的免疫荧光信号首先出现在原肠胚期多个细胞的细胞膜上，然后出现在担轮幼虫期的肠上，在随后的发育阶段中，CgTAR1的免疫荧光信号主要出现在长牡蛎幼虫的面盘、壳顶和鳃等处，这表明CgTAR1在长牡蛎幼虫感应外界环境等生理过程中可能也扮演着一定的角色。在长牡蛎的所有成体检测组织中，均检测到CgTAR1的mRNA转录水平。其中，CgTAR1在性腺中的转录水平最高，在消化腺中的转录水平最低，这表明CgTAR1可能在长牡蛎的成体组织中也发挥着一定的作用。我们对CgTAR1的研究为后续进一步地研究CgTAR1及其近源蛋白在海洋贝类中的生理功能奠定了良好的基础。
其他摘要	G protein-coupled receptors (GPCRs) compose a large family of membrane receptors and are considered as the largest family of cell-surface receptors. As the mediated moleculars, GPCRs transduct extracellular signals into cells via binding of G protein. GPCRs are involved in various important physiological activities, including sense of sight, taste and smell, regulation of emotion, control of behavior, regulation of nervous system, endocrine system and immune system. Therefore, GPCRs have been widely used in pharmaceutical industry as the target molecular for drugs. Most of the GPCRs belong to the Rhodopsin family (Class A) in living organisms. In the Rhodopsin family, biogenic amine receptors, such as octopamine (OA) receptor, tyramine (TA) receptor, dopamine (DA) receptor, epinephrine receptor and serotonin receptor, are involved in various important vital movements in both vertebrates and invertebrates. Thus, biogenic amine receptors have gained considerable attention from many scientists. Biogenic amine receptors can bind with biogenic amines. They exist in plants and animal, especially in many kinds of food. The molecular weight of biogenic amine is usually not very high. As the putative neurotransmitter, biogenic amines are involved in various important physiological activities, including growth, maturation, and reproduction. OA and its precursor TA are common biogenic amines in invertebrates. They are involved in numerous developmental process of invertebrates such as reproduction, maturation and metamorphosis, and play very important role in these vital process. Although OA was first discovered in octopus (mollusks), followed studies on OA, TA and related receptors have primarily been conducted in Ecdysozoa, especially in insects. While in mollusks such as bivalve molluscs, only limited reports are available and their physiological roles remain unclear. In this dissertation, we mainly conducted the physiological functional characterization of OA/TA during different developmental stages of the Pacific oyster, Crassostrea gigas. Ⅰ*. Classification and expression profile analysis of GPCRs in C. gigas.* Based on the complete genome sequences of C. gigas previously obtained from our research group, about 553 putative GPCRs were screened, identified and annotated from C. gigas. Subsequently, via hydrophobic transmembrane area analysis and family domain screening, 412 GPCRs were isolated and identified. By means of BLAST homologous alignment and phylogenetic analysis, the classification of these sequences was finally determined. Among these sequences, 308 GPCRs were classified as Class A; 44 GPCRs were classified as Class B; 14 GPCRs were classified as Class C and 46 GPCRs belong to the unknown family of GPCR protein. In addition, by statistical analysis of the Class A GPCRs in C. gigas, at least 36 GPCRs belong to the subfamily of biogenic amine receptors. Subsequently, via expression profile analysis from different developmental stages of C. gigas, the expression patterns of these GPCRs were classified into 4 types: (1) no expression or very low level of expression for GPCRs was detected during all the developmental stages of C. gigas; (2) GPCRs were mainly expressed in the early developmental stages of C. gigas; (3) GPCRs were mainly expressed in the mid-developmental stages of C. gigas; (4) GPCRs were specificly expressed in some developmental stages of C. gigas. Notably, 2 novel biogenic amine receptors (CGI_10017568 and CGI_10027100) were also identified from the fourth type, which were considered to involve in the metamorphosis process of C. gigas. Ⅱ. Functional Characterization of novel OA/TA receptor CgGPR1 *during different developmental stages of C. gigas.* Via PCR and RACE, a full-length cDNA of 2,826 bp and encoding a putative 524 amino acid OA/TA receptor (CgGPR1, CGI_10017568) was isolated from C. gigas. According to the BLAST results, CgGPR1 was most closely related to the Lymnaea stagnalis OA receptor OAR2 in sequence. Phylogenetic analysis showed that CgGPR1 belongs to a poorly studied subfamily of invertebrate OA/TA receptors. Subsequently, the spatio-temporal expression of CgGPR1 in different developmental stages and adult tissues of C. gigas was examined by quantitative real-time PCR, Western Blot and whole-mount immunoreactive fluorescence analysis. The results showed that CgGPR1 was expressed during all developmental stages of C. gigas with higher levels at the gastrula stage, trochophore stage and D-shaped larva stage, indicating its potential role in embryogenesis and tissue differentiation. Immunoreactive fluorescence of CgGPR1 was mainly observed in the velum, foot, gill and mantle of C. gigas larvae, indicating its potential role in the swimming, filter-feeding and responding to external environment of C. gigas larvae. Immunoreactive fluorescence of CgGPR1 was also observed in the foot of pediveliger larva stage, indicating its potential role during the metamorphosis of C. gigas. CgGPR1 transcripts were detected in all the tested organs of adult C. gigas, with highest level in the mantle and lowest level in the gonad, which suggested that CgGPR1 may also play roles in the the organs of adult C. gigas. Moreover, by establishing HEK293 cell lines that could stably express CgGPR1, the influence of CgGPR1 to the level of cAMP and Ca²⁺ in HEK293 cells expressing CgGPR1 was conducted with biogenic amine agonists. Pharmacological analysis indicated that cAMP and Ca²⁺ concentrations remained unchanged in HEK293 cells expressing CgGPR1 upon addition of OA, TA or related amines, implying that CgGPR1 uses other unknown molecules as second messenger rather than cAMP and Ca²⁺. Our study lays the foundation for further research of the physiological function of CgGPR1 and its homologous proteins in invertebrates, and provides a potential target molecular for the prevention of marine fouling. Ⅲ. Spatio-temporal expression pattern analysis of novel TA receptor CgTAR1 *during different developmental stages of C. gigas.* A full-length cDNA of 1,601 bp and encoding a putative 407 amino acid TA receptor (CgTAR1, CGI_10027100) was also isolated from C. gigas. According to the BLAST results, CgTAR1 was most closely related to the Locusta migratoria TA receptor TAR1 in sequence. Phylogenetic analysis showed that CgTAR1 belongs to the α₂- adrenergic like tyramine subfamily of invertebrate OA/TA receptors. Afterwards, the spatio-temporal expression of CgTAR1 in different developmental stages and adult tissues of C. gigas was examined by quantitative real-time PCR, Western Blot and whole-mount immunoreactive fluorescence analysis. The results showed that CgTAR1 transcripts were highly expressed in the morula stage, blastula stage and pediveliger larva stage and poorly expressed in the D-shaped larva stage and umbo larva stage. While at the protein level, CgTAR1 was highly expressed from the egg stage to D-shaped larva stage, indicating its potential role in embryogenesis, tissue differentiation and organ formation process of C. gigas. Immunoreactive fluorescence of CgTAR1 was firstly observed in the membrane of many cells in the gastrula stage of C. gigas, and then in the gut of the trochophore stage. Subsequently, immunoreactive fluorescence of CgTAR1 was mainly observed in the velum, umbo and gill, indicating its potential role in the responding to external environment of C. gigas larvae. CgTAR1 transcripts were detected in all the tested organs of adult C. gigas, with highest level in the gonad and lowest in the digestive gland, which indicated that CgTAR1 may also play roles in the the organs of adult C. gigas. Our study sheds light on the physiological function of CgTAR1 in oysters.
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136536
专题	实验海洋生物学重点实验室
作者单位	1.中国科学院海洋研究所 2.中国科学院大学
推荐引用方式 GB/T 7714	纪鹏. 生物胺类受体在长牡蛎不同发育时期中的功能研究[D]. 北京. 中国科学院大学,2017.

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