【作者】 郭斌；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 硕士

【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。弗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。本论文对文昌鱼(Branchiostoma belcheri) IGF基因的克隆、表达、功能及其受GH作用上调表达展开了研究,从而提供了文昌鱼存在GH/IGF轴的证据。GH/IGF轴在所有的脊椎动物中都存在,但它的进化起源还不清楚。我们从头索动物文昌鱼中克隆并鉴定了一个编码IGF多肽的cDNA。我们克隆得到的cDNA全长为1630 bp,开放阅读框(ORF)为924 bp,5’非编码区长23 bp,3’非编码区长683 bp。ORF编码一个307个氨基酸的蛋白质,BbIGF,其理论分子量为35 kDa,在23-101位上具有IGF_insulin_like的保守结构域。BbIGF前101位包括了24位的信号肽,B链,C链和A链,具有胰岛素前体的结构特点;后面部分又可分为D链和E链,这又是胰岛素样生长因子的结构特点。在BbIGF的B链和A链中,我们找到了六个保守的用于形成链内和链间二硫键的半胱氨酸残基。Southern杂交表明BbIGF在青岛文昌鱼基因组中是单拷贝的,这与加利福尼亚文昌鱼的insulin/IGF基因为单一拷贝结果一致。Northern blot显示BbIGF只有一个转录产物,长度约为2100bp。切片原位杂交结果显示,文昌鱼的BbIGF基因在肝盲囊和后肠特异性表达。这也为免疫组织化学的结果所证实,BbIGF主要位于肝盲囊和后肠的细胞质中。BbIGF的表达模式为文昌鱼肝盲囊与脊椎动物肝脏同源的假说提供了论据。此外,免疫印迹实验表明BbIGF在文昌鱼体液中也存在,提示在消化系统中合成的多肽能够被分泌到血液中,经血液循环流遍整个身体。将阳性重组表达质粒pET32a/BbIGF转化大肠杆菌BL21感受态细胞后,用IPTG诱导表达,用镍离子亲和树脂进行纯化。运用MTT法检测FG-9307细胞的生长情况,结果表明体外表达的重组BbIGF蛋白具有促有丝分裂的功能,能促进牙鲆鳃细胞的增殖。但重组BbIGF不能降低血糖水平,提示它可能不具有胰岛素的功能。以上研究表明BbIGF在功能上与IGF更为相似。从文昌鱼中剥取肝盲囊,切碎(1 mm3),在加入了100 ng/ml和0 ng/ml(对照)的重组鼠GH的培养基中培养。实时定量PCR结果显示,外源重组鼠GH对BbIGF在文昌鱼肝盲囊中的表达有显著的促进作用。此外,GHR是一种存在于脊椎动物肝脏里的整合膜蛋白,RIPA是适用于提取膜蛋白的缓冲液。通过Western blotting技术发现文昌鱼肝盲囊存在一个类似鼠GHR的膜蛋白分子。这些研究结果都表明在文昌鱼中GH和IGF存在密切的联系,由此将GH/IGF轴进化的源头向前推进到了原始的脊索动物,也为进一步研究原索动物的GH/IGF轴铺平了道路。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, expression, function of amphioxus IGF and its up-regulation by GH, demonstrating GH/ IGF axis in amphioxus Branchiostoma belcheri.The GH/IGF axis is unique to all vertebrate species, but its evolutionary origin is ill-defined. In this study, we have cloned and characterized the cDNA of an IGF polypeptide in amphioxus B. belcheri. The cDNA obtained was 1630 bp long with an open reading frame (ORF) of 924 bp, a 5′-untranslated region (UTR) of 23 bp and a 3′-UTR of 683 bp. The ORF encoded a polypeptide of 307 amino acids with a molecular mass of approximately 35 kDa. The deduced long polypeptide, BbIGF, has an IGF_insulin_like domain at residual positions 23-101. The first one-third (residues 1-101) of the polypeptide was organized much like a typical preproinsulin with a signal peptide (24 residues), B chain, C-peptide and A chain, whereas its remaining sequence (residues 102-307) could be divided into putative D and E domains reminiscent of proIGF. Six conserved cysteine residues required for the formation of inter- and intra-chain disulphide bonds were identified in the B and A domains of BbIGF. Southern blotting demonstrates that BbIGF is a single copy gene, which is in line with that of B. californiensis insulin/IGF gene. Northern blotting revealed the presence of a single band of about 2.1 kb transcript in B. belcheri. In situ hybridization histochemistry showed that BbIGF transcript was specifically expressed in the hepatic caecum and hind-gut. This was further corroborated by the immunohistochemical staining, showing that BbIGF was localized in the cytoplasm of hepatic caecum and hind-gut. This basically agrees with the hypothesis that amphioxus hepatic caecum is homologous to the vertebrate liver in respect to IGF expression pattern. Also, the presence of BbIGF in the humoral fluids, as evidenced by immunoblotting, suggests that the peptide synthesized in the digestive system can be secreted into the blood, circulating via blood stream throughout the body.An expression vector including the cDNA coding for mature BbIGF and 5’additional tags of pET32a was constructed and transformed into E. coli cells. The recombinant peptide was induced by IPTG and purified by affinity chromatography on a Ni-NTA resin column. The mitogenic effect of BbIGF was examined by measuring the growth rate of FG-9307 cells via MTT method. The recombinant BbIGF expressed in vitro was capable of stimulating the flounder gill cell proliferation. Unexpectedly, BbIGF was not able to reduce blood glucose level, suggesting that it had little insulin activity. This suggests that BbIGF is functionally more related to IGF rather than insulin.The hepatic caecums were dissected out of amphioxus B. belcheri, cut into pieces (1 mm3) and cultured in the medium plus 100 ng/ml and 0 ng/ml (control) of the rat recombinant GH. Quantitative real-time PCR demonstrated that exogenous recombinant rat GH was able to induce a significant up-regulation of BbIGF expression in the hepatic caecum. Moreover, GHR was an integral membrane protein in the vertebrate liver, and RIPA was a suitable buffer for the extraction of this molecule. Western blotting revealed the presence of a membrane protein molecule similar to rat growth hormone receptor in the hepatic caecum. These apparently establish a definitive link between GH and IGF in B. belcheri, pushing the evolutionary origin of GH/IGF axis to the primitive chordate. These also pave the way for further characterization of the GH/IGF axis in the protochordate.更多还原