【作者】 张敏；

【导师】 刘振辉；

【作者基本信息】 中国海洋大学 ， 发育生物学， 2012， 硕士

【摘要】 B类清道夫受体(theclassBscavengerreceptor,SR-B)是一种位于细胞表面的跨膜糖蛋白，主要包括SR-BI、SR-BII和CD36。SRB可与多种配基结合，其在细胞和组织中分布也较为广泛，参与很多生理和病理过程，如机体免疫防御、凋亡细胞清除、动脉粥样硬化及其它心血管疾病等。目前在很多物种中SR-B基因的序列、结构、表达和功能已经得到了比较深入的研究。而在文昌鱼中的研究却一直未见报道。文昌鱼是介于无脊椎动物和脊椎动物之间的过渡类型，是研究脊椎动物起源和进化的模式动物。本文克隆了青岛文昌鱼(Branchiostomajaponicum）的具有完整开放阅读框的SR-B同源基因(BjCD36)，并对其进化、表达和功能进行了研究。主要研究结果如下：第一，我们对青岛文昌鱼中的SR-B基因进行了鉴定。在弗罗里达文昌鱼中我们发现了五个SR-B的同源基因(SRB-a,b,c,d,e)，它们编码的氨基酸序列长度分别是480aa、445aa、912aa、229aa、159aa，但是通过NCBI上保守的结构域分析发现只有三个基因(SRB-a,b,c）编码的蛋白质有完整的CD36结构域。最后通过多序列比对发现，SRB-a与SR-B的相似度与其他四个相比更高，并且SRB-a与CD36的相似度比它与SR-BI/II的相似度要高，另外，通过基因结构分析发现，SRB-a基因的基因结构与脊椎动物尤其是斑马鱼的极为相似。所以我们首先将SRB-a基因确定为我们在青岛文昌鱼中的研究对象。此外，我们对脊椎动物CD36基因的基因结构、内含子类型和在染色体上的位置等进行了分析，结果显示CD36基因在脊椎动物中高度保守，暗示CD36基因的功能在进化过程中可能具有保守性。第二，我们在青岛文昌鱼中通过RACE-PCR技术克隆了SRB-a（本文命名为BjCD36）并对其序列进行了分析。BjCD36全长1711bp，开放阅读框(ORF)含有1482bp，编码494个氨基酸的蛋白质。并且通过多序列比对发现该序列与弗罗里达文昌鱼中SRB-a基因的氨基酸序列之间的相似性高达90%。第三，我们对BjCD36基因在文昌鱼中的组织和胚胎发育中的时空表达情况做了研究。Real-timePCR和切片原位杂交结果显示，BjCD36在成体组织的肝盲囊中表达量最高，在其他组织中的表达相对要低。胚胎原位杂交显示，BjCD36在早期胚胎及一天幼虫中都有表达，在两天幼虫中是在幼虫的前三分之一处表达，表明该基因可能在胚胎发育中发挥重要作用。第四，为了研究BjCD36基因是否有免疫相关功能，我们用大肠杆菌处理文昌鱼，发现大肠杆菌感染后BjCD36的表达上调。此外，我们用不同浓度的LTA处理转染了BjCD36的牙鲆鳃细胞系(FG)，结果发现10μg/ml或20μg/ml的LTA都能诱导BjCD36上调表达。体内和体外实验都证明了BjCD36是与免疫相关的基因，参与到机体免疫过程。为了研究BjCD36是否是与摄食调控相关，我们对文昌鱼先喂食，接着进行饥饿处理，发现在喂食后2h鳃中BjCD36的表达显著升高，随后随着饥饿时间的延长，BjCD36的表达逐渐降低，表明BjCD36基因确实是参与到了文昌鱼的摄食调控过程中。由此，我们证明BjCD36参与免疫与摄食调控，与高等脊椎动物CD36基因具有功能上的保守性。更多还原

【Abstract】 Scavenger receptor class B (SRB) are transmembrane glycoproteins and contain three members:CD36, SR-BⅠ and SR-BⅡ. SRB can bind a wide variety of ligands and express on many cells and tissues, so they can mediate diverse normal physiological and pathological processes, such as innate immune defence, internalization of apoptotic cells, atherothrombotic diseases and other cardiovascular diseases. The structures, functions, sequences and expressions of many kinds of SR-B have been studied, yet little is known to date about it in the amphioxus Branchiostoma japonicum. Amphioxus or lancelet, a cephalochordate, located between the invertebrates and vertebrates, and is becoming an emerging model organism for insights into the origin and evolution of vertebrates. In this paper, we report cloning, evolution, expression and functional analysis of BjCD36gene in amphioxus Branchiostoma japonicum. The main results are as follows:1Five SR-B homologues (SRB-a, b, c, d, e) were identified in amphioxus Branchiostoma floridae, and they encoded proteins with length of480,445,912,229and159amino acids, respectively, but only theree of them (SRB-a, b, c) contained the CD36domain through the analysis of Conserved Domain Database in NCBI. The alignment of multiple protein sequences among SR-B revealed that the SRB-a was more similar to SR-B than other four genes and the similarity between SRB-a and CD36is more high than SRB-a and SR-BⅠ/Ⅱ. Also, analysis of exon-intron organization showed that the exon-intron organization of SRB-a was very similar to the that of vertebrates (especially zebrafish). So the SRB-a gene was indentified as the homology of CD36. Moreover, analysis of the exon-intron organization, the intron type and the chromosome locus revealed that CD36gene was conserved during evolution. 2The cDNA encoding SRB-a was obtained by RACE-PCR in amphioxus B. japonicum, and was named BjCD36in this paper. The full-length cDNA of BjCD36was1711bp long, containing an open reading frame (ORF) of1482bp which encoded a protein of494amino acids. BjCD36showed90%of sequence identity with SRB-a from amphioxus B.floridae in amino acid level.3Real-time PCR and in situ hybridization demonstrated that BjCD36transcript was abundant in hepatic caecum, and at a slightly lower level in other tissue. Whole mount in situ hybridization showed that BjCD36transcript was widely expressed in the early embryos and the1-day larvae, but was expressed in the front one third of the full-length body in2-day larvae, where the gill and hepatic caecum will form later during development.4Real-time PCR indicated that challenge with E. coli (0h-48h) resulted in an up-regulation of BjCD36expression in the gut-free bodys of amphioxus, and10μg/ml or20μg/ml of LTA-induced BjCD36up-regulation in the FG cells transfected with BjCD36was found. These results suggested that BjCD36may be involved in the host immune defense. In order to detect the response of BjCD36F-a to feeding, the adult amphioxus was fed with algae S. platensis, then was challenged with hungry. It was found that BjCD36was up-regulated after feeding, then a down-regulation of BjCD36expression in the gills was decteced with hungry challenge, indicating the roles of BjCD36in the feeding control. Together these studies identify and characterize a conserved gene that is important in the fundamental process of immune and feeding regulation.更多还原