【作者】 蒋圣娟；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2007， 博士

【摘要】 文昌鱼是介于无脊椎动物和脊椎动物之间的过渡类型,是现存的与脊椎动物祖先最接近的无脊椎动物,一直被作为研究生物进化和胚胎发育的典型材料。佛罗里达文昌鱼基因组的测序完成,为利用分子生物学手段,研究文昌鱼基因的结构、进化和表达,揭示文昌鱼发育的分子生物学机制奠定了坚实的基础。本论文对青岛文昌鱼(Branchiostoma belcheri tsingtaunese) TRP14和ycaCR基因的克隆与鉴定、结构、进化、表达以及功能展开了研究。AmphiTRP14,是硫氧还蛋白相关蛋白家族成员,全长775 bp,最大开放阅读框编码由123个氨基酸组成的蛋白质,其理论分子量大约为14 kDa,理论等电点为4.8。该蛋白质与人的TRP14蛋白的相似性为56%,序列中含有一个类似Trx的基序Cys-Pro-Asp-Cys (CPDC),二级结构含有五个β折叠和五个α螺旋,这些都是TRP14蛋白的典型特征。高等生物(人、动物和植物)到细菌体内都存在着TRP14同源序列。对TRP14蛋白同源序列和代表性的硫氧还蛋白序列,通过neighbor-joining方法构建的系统进化树显示:AmphiTRP14蛋白和人类的TRP14以及其他物种的TRP14蛋白聚到一起,而和Trx蛋白归属不同分支,这表明TRP14蛋白是一类含有CPDC序列,不同于Trx的保守蛋白。从佛罗里达文昌鱼基因组数据库中也搜索到TRP14,和青岛文昌鱼TRP14同源性达94%,其基因组序列包含四个外显子和三个内含子,与后口动物包括人、牛、大鼠、小鼠和海胆的TRP14基因结构一致。原口动物如果蝇和线虫TRP14基因则含有三个外显子和两个内含子。这显示TRP14基因four-exon-three-intron的结构在原口动物和后口动物分化后出现,并且在后口动物中高度保守。青岛文昌鱼的TRP14基因插入pET28a融合表达载体,在原核E.coli中实现了高效稳定的表达,并纯化出AmphiTRP14融合蛋白。用还原胰岛素二硫键的方法,发现AmphiTRP14蛋白具有还原酶的活性,并且显示出一定的浓度依赖性。AmphiTRP14蛋白的二硫键的还原能够将色氨酸的荧光强度提高近2.7倍,这暗示色氨酸残基周围微环境的变化。切片原位杂交结果显示,AmphiTRP14在肝盲囊、卵巢、鳃和肠表达丰度较高,而在其他组织中表达较低或者是没有表达,表明TRP14在文昌鱼体内的表达具有组织特异性。利用AmphiTRP14原核表达重组蛋白质制备的多克隆抗体进行免疫组织化学杂交,发现在蛋白水平也存在类似的表达分布,而人类TRP14蛋白分布于所有组织内,呈现广泛表达的模式,这说明AmphiTRP14蛋白在这些组织的高表达可能和其易于被氧化压力的损害有关或者是在食物消化过程中起特殊的作用。本文还报道一个类ycaC的基因AmphiycaCR,这是在除大肠杆菌以外的物种中首次报道。它全长891 bp,最大开放阅读框编码由201个氨基酸组成的蛋白质,其理论分子量大约为22KDa,理论等电点为7.2。AmphiycaCR含有一个保守的ycaC-related的蛋白质结构域,且含有保守的可能的催化残基D19、R (K) 84和C118(1YAC;按大肠杆菌ycaC中标号)和保守的非脯氨酸的顺式肽键,并且具有与ycaC蛋白相类似的三层三明治拓扑结构,这是ycaC家族的典型特征。系统进化分析发现,AmphiycaCR蛋白单独聚类,与Isochorismatase超家族的其他亚家族明显分开,说明AmphiycaCR与ycaC虽然有类似的残基,但分属不同的亚家族。从佛罗里达文昌鱼基因组数据库中也搜索到ycaCR基因,和青岛文昌鱼ycaCR氨基酸同源性达93%,其基因组序列包含四个外显子和三个内含子,后口动物包括人、大鼠和狗的ycaCR基因结构含有五个外显子和四个内含子,原口动物如果蝇和线虫ycaCR基因则含有三个外显子和两个内含子,说明在物种进化过程中,该基因外显子呈现增多趋势。应用原核蛋白表达的方法构建原核表达载体,纯化融合蛋白AmphiycaCR。用Isochorismatase,Nocitinamidase和N-carbamoylsarcosine amidohydrolase活性测定方法测定AmphiycaCR的活性,发现AmphiycaCR不具有这三种酶的活性。蛋白质互作实验鉴定了两种相互作用蛋白,一个是文昌鱼组织液中正常含有的ycaCR蛋白,说明文昌鱼ycaCR蛋白可能是以同源多聚体发挥生理功能;另一个是肌酸激酶,可能通过相互结合间接参与能量反应。AmphiycaCR切片原位杂交表明AmphiycaCR在成体文昌鱼不同组织中表达量变化较大,在肝盲囊、肠、内柱和卵巢表达量较高,其他组织表达量较低,呈现完全表达模式。制备多克隆抗体,并应用于免疫组织化学定位,研究结果与切片原位杂交结果一致。结果说明这是一类位于线粒体的isochorismatase超家族成员,可能与肌酸激酶相互作用。广泛表达的模式说明它可能行驶基本的生理功能,肝盲囊、后肠、卵巢和内柱的高量表达与这些组织的快速能量代谢有关。更多还原

【Abstract】 Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. It is well-known model organism widely used for interspecies comparative genome studies and developmental homology analysis. Sequencing of the whole genome of Florida amphioxus offers a new opportunity to the research of molecular and evolutionary biology of amphioxus. In this paper, we report the cloning, characterization, expression, phylogenetic analysis and functional characterization of amphioxus TRP14 gene, AmphiTRP14 and amphioxus ycaC-related gene, AmphiycaCR from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtaoensis.AmphiTRP14 obtained from the gut cDNA library of amphioxus B. belcheri is 775 bp long and its longest open-reading frame (ORF) codes for a protein of 123 amino acids with a predicted molecular mass of approximately 14 kDa and an isoelectric point of 4.8. The deduced amino acid sequence shares 56% identity to human TRP14, and had a Trx-like motif Cys-Pro-Asp-Cys (CPDC). It has fiveβ-sheets and fiveα-helixes, characteristic of human TRP14. There are a number of putative TRP14 sequences from a variety of organisms including animals, plants and bacteria. The phylogenetic tree constructed by neighbor-joining method using the sequences of TRP14 proteins, putative TRP14 proteins and representative Trx proteins shows that both AmhiTRP14 and human TRP14 as well as putative TRP14 proteins are clustered together, separating from Trx proteins. These imply that TRP14 is a highly conserved ubiquitous protein with the active site CPDC. A search of the recently completed draft assembly and automated annotation of B. floridae genome reveals the presence of a Florida amphioxus TRP14 cDNA and its genomic DNA sequence. Sequence comparison demonstrates that AmphiTRP14 shares 94% identity to the deduced protein encoded by Florida amphioxus gene at the amino acid level. Analysis of the genomic structure exhibites that Florida amphioxus TRP14 gene consists of four exons and three introns, which is in accord with the genomic structures from deuterostomes such as human, bovine, mouse, rat and sea urchin, while putative TRP14 genes in prostostomes like fruit fly and nematode comprise three exons and two introns. These demonstrate the evolutionary emergence of the four-exon-three-intron organization of TRP14 genes after the split of protostome/deuterostome, which is highly conserved since then. An expression vector including the entire open reading frame of AmphiTRP14 and a 5’additional tag of pET28a is constructed and transformed into E. coli. Recombinant protein is expressed and purified. AmphiTRP14, which contains W40, W75 and W99, yielded a 2.7-fold increase in tryptophan fluorencence when reduced, suggesting changes in the microenvironment around the Trp (W) residues juxtaposing the CXXC active site. AmphiTRP14 exhibits a concentration-dependent disulfide reductase activity toward insulin, which contains one intramolecular and two intermolecular disulfides. It shows that AmphiTRP14 is a disulfide reductase. In situ hybridization histochemistry reveals that AmphiTRP14 transcript is abundant in the hepatic caecum and ovary, and at a lower level present in the hind-gut, endostyle, epipharyngeal groove, gill and testis, while it is absent in the neural tube, notochord and muscle. It is clear that TRP14 gene is expressed in amphioxus in a tissue-specific manner. This is further corroborated by immunohistochemical staining using the rabbit antiserum against the purified recombinant AmphiTRP14, which showed that AmphiTRP14 was predominantly localized in the hepatic caecum, ovary and hind-gut, and weakly in the testis and gill. This suggests that AmphiTRP14 may play a fundamental but tissue-specific role in food digestion for example, or alternatively reflect differences in the tissue susceptibility to oxidative damage.This paper also reports the characterization and expression of AmphiycaCR. The cDNA is 891 bp long and the ORF codes for a protein of 201 amino acids with a predicted molecular mass of approximately 22 kDa and an isoelectric point of 7.2. The deduced protein has a ycaC related domain, which belongs to the the isochorismatase superfamily. Like ycaC, AmphiycaCR had the putative catalytic triad: D19, R(K)84 and C118 (numbering as in E.coli) and the predicted structure has three-layerα-β-αsandwich topology structure, characteristic of the ycaC family. The phylogenetic tree constructed by neighbor-joining method using the sequences of putative ycaCR proteins and representative members of the isochorismatse superfamily shows that AmphiycaCR and other putative ycaCR proteins are clustered together, separating from other subfamilies of the isochorismatase superfamily. These imply that ycaCR is a highly conserved protein with the active sites of ycaC subfamily. An expression vector including the entire ORF of AmphiycaCR and a 5’additional tag of pET28a is constructed and transformed into E. coli. Recombinant protein is expressed and purified. Assays for isochorismatase, nicotinamidase and N-carbamoylsarcosine amidohydrolase demonstrate that AmphiycaCR doesn’t show the activity of the three enzymes. Pull down assay shows that there are two proteins interacting with AmphiycaCR. One is amphioxus native ycaCR protein and the other is creatine kinase. These showed that AmphiycaCR may be form polymers and indirectly involved in energy transduction. In situ hybridization histochemistry reveals that AmphiycaCR transcript is abundant in the hepatic caecum, gut, endostyle and ovary, and at a lower level present in the epidermis, epipharyngeal groove, gill, testis, muscle, neural tube and notochord. It is clear that AmphiycaCR gene is expressed in a ubiquitous expression pattern. This is further corroborated by immunohistochemical staining using the rabbit antiserum against the purified AmphiycaCR, which shows that AmphiycaCR is predominantly localized in the hepatic caecum, endostyle, ovary and hind-gut, and weakly in the testis and gill. This suggests that AmphiycaCR may be a novel member of isochoristamase superfamily, which is mainly localized in the mitochondrial fraction, and is capable of interacting with the creatine kinase. The ubiquitous expression pattern suggests that Amphi plays a conserved fundamental cellular role in amphioxus, lending additional support to the proposal that Amphi is involved in energy transduction. The predominant expression in the gill, hepatic caecum, hind-gut, endostyle, and ovary may be in line with a rapid energy turnover in these tissues.更多还原