【作者】 王韶辉；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 文昌鱼(Amphioxus)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,一直被认为是研究脊椎动物起源和进化的重要模式动物。甲状腺-肝脏轴在包括七腮鳗在内的脊椎动物中普遍存在,然而其起源还是一个谜。在文昌鱼中有一个与甲状腺同源的器官——内柱,有一个肝脏同源的器官——肝盲囊,暗示文昌鱼中可能存在类甲状腺-肝脏轴,但是,尚没有证据证明文昌鱼内柱(类甲状腺)和肝盲囊(类肝脏)之间存在功能上的联系。本论文的主要部分就是论证文昌鱼中存在类脊椎动物甲状腺-肝脏轴信号通路。本论文首先从文昌鱼中克隆到一个肝富集转录因子CAAT区/增强子结合蛋白BbC/EBPα/β的开放阅读框,可编码—80个氨基酸的蛋白质序列,分子量为9.3kDa。该蛋白序列同其它物种C/EBPα和C/EBPβ氨基酸序列进行比对分析,BbC/EBPα/β氨基酸序列与C/EBPα的相似性为51.9-54.4%,与C/EBPβ的相似性为51.9-59.7%。这说明BbC/EBPα/β与C/EBPα和C/EBPβ具有相同的相似性,因而本论文将其命名为BbC/EBPα/β。它与从弗罗里达文昌鱼基因组文库中分析出来的C/EBP的相似性达到94.3%。本论文推测BbC/EBPα/β可能是脊椎动物C/EBPα和C/EBPβ的原型。这个结论为构建的系统进化树进一步证实。构建的进化树表明BbC/EBPα/β蛋白位于脊椎动物C/EBPα和C/EBPβ分枝的基部,而C/EBPα和C/EBPβ各自聚群。组织特异性分析表明,BbC/EBPα/β同脊椎动物的C/EBPα和C/EBPβ一样,主要在肝盲囊表达。本论文又克隆了文昌鱼的甲状腺激素受体BbTR基因的完整开放阅读框,可编码一430个氨基酸的蛋白质序列,其分子量为49 kDa。BbTR基因推导的氨基酸序列与已报道的弗罗里达文昌鱼的TR相似性高达94.3%,且主要活性位点保守。组织特异性分析表明,BbTR在包括肝盲囊在内的全身各处都有表达。原位杂交结果表明,BbTR在内柱中的表达主要集中在碘结合区(5区和6区),佐证了内柱与甲状腺的同源。实时定量PCR结果表明,不管是在体内还是体外实验中,甲状腺激素(T4)、三碘甲腺原氨酸(T3)和它们的衍生物三碘甲腺乙酸(TRIAC)均能刺激BbC/EBPα/β在肝盲囊中的表达,但是T4的作用相对要弱一些。而且,T4的作用可以被脱碘抑制剂碘泛酸抑制,说明T4要在体内脱碘转换成T3才能发挥作用。荧光猝灭实验表明T3和TRIAC都能和重组表达的BbTR结合,而T4不能结合。另外,本论文检测到内柱中富含T3和T4,同脊椎动物的甲状腺一样。结果说明,在文昌鱼中可以通过T3/TRIAC-TR的方式诱导BbC/EBPα/β表达。也就是说,文昌鱼中可能存在类脊椎动物甲状腺-肝脏轴的信号通路。本文第二部分是重组表达斑马鱼卵黄蛋白原(Vg)的卵黄高磷蛋白(Pv)部分,并对其抑菌活性进行研究,推测其在胚胎发育中的功能。首先,本论文重组表达了斑马鱼的Pv,并进行了质谱鉴定。然后,本论文检测了Pv的抑菌活性,发现Pv对大肠杆菌和金黄色葡萄球菌都有很强的抑制作用,电镜扫描结果发现菌体表面受到破坏。将异硫氰酸荧光素(FITC)标记的重组Pv与各种菌孵育后荧光显微镜观察发现重组PV可以分别与E. coli和S. aureus结合；另外通过酶联免疫吸附实验(ELISA)证明重组Pv可以与病原相关模式分子(PAMPs)脂多糖(LPS)、脂磷壁酸(LTA)、肽聚糖(PGN)发生特异性结合,并且结合呈现浓度依赖性。结果表明Pv是一种多价的模式识别受体。铁结合实验表明重组Pv不能与铁结合,添加铁离子也不能减弱重组Pv的抑菌活性。这表明重组Pv的抑菌活性与夺铁机制无关。ELISA实验检测Pv在胚胎发育不同时期的含量结果显示,在胚胎发育初期,含有较高的Pv浓度,随着发育时间的延长,Pv含量逐渐减少,但直到12小时胚胎内Pv浓度仍维持在可以显著抑菌水平之上。这些结果说明在发育早期,Pv起着营养作用以外的免疫防御功能。为寻找Pv的抑菌活性中心,本论文先采用氨基端切除的方法,发现直到切除了氨基端194个氨基酸残基,剩下的55个氨基酸残基仍有抑菌活性。在此基础上,采用定点突变的方法,继续寻找抑菌活性中心。结果显示,带正电荷和疏水的氨基酸残基对Pv的抑菌活性起到关键的作用。总而言之,Pv在斑马鱼胚胎发育过程中起着免疫防御的功能,而不仅仅是营养物质。Pv的抑菌活性中心在羰基端,带正电荷和疏水的氨基酸残基对于抑菌活性比较关键。更多还原

【Abstract】 The thyroid-liver axis is present in all the vertebrates including lamprey. Amphioxus, a primitive chordate, has been shown to possess a thyroid-like organ, endostyle, as well a liver-like organ, hepatic caecum, suggesting the presence of a vertebrate-like thyroid-liver axis in this animal. However, a definitive link between these organs remains to be established. This study mainly dealt with the demonstration of the presence in amphioxus of a signaling flow from the endostyle to the hepatic caecum.The cDNA of BbC/EBPα/βobtained contained an open reading frame (ORF) of 240 bp encoding a deduced polypeptide of 80 amino acids with a molecular weight of 9.3 kDa. Sequence comparison revealed that the polypeptide of BbC/EBPα/βshared 94.9% identity to B.floridae C/EBP and was 51.9-54.4% and 51.9-59.7% identical to the vertebrate C/EBPa and C/EBPβ, respectively. It was clear that BbC/EBPa/p was closely equally similar to C/EBPαand C/EBPβin sequence (therefore named C/EBPα/β), suggesting that BbC/EBPα/βmay be the archetype of vertebrate C/EBPa and C/EBPβ. This was also supported by the phylogenetic tree constructed which showed that C/EBPa and C/EBPP were each grouped together, forming two separated clades, while BbC/EBPα/βwas located at the base of both C/EBPa and C/EBPP clades. qRT-PCR showed that BbC/EBPα/βtranscript was most abundant in the hepatic caecum, which strengthens the notion that the hepatic caecum is homologous to the vertebrate liver. The BbTR cDNA obtained was 1317 bp long with an ORF of 1290 bp encoding a deduced protein of 430 amino acids with amolecular size of about 49 kDa. Sequence comparison revealed that BbTR shared 94% identity to B. floridae TR peptide. BbTR was found to be widely expressed in all the tissues examined, including the hepatic caecum, but it was abundantly expressed in the endostyle. In agreement, in situ hybridization histochemistry also revealed that BbTR was mainly expressed in the endostyle and gill. In the endostyle, BbTR was predominantly expressed in the zones 5a and 5b of the endostyle and at a lower level present in the zones 6 and 1 of the tissue. The zones 5 and 6 had been shown to be the iodine-binding center, and therefore our results showed that in the endostyle, the cells expressing BbTR were basically restricted in the iodine-binding regions.Both in vivo and in vitro assays showed that the expression of BbC/EBPα/βin the hepatic caecum was significantly increased following the exposure to T3,T4 or TRIAC. but compared with T3 and TRIAC, T4 acts less effective. And IOP, a deiodinase inhibitor, was able to inhibit T4 action, whereas T3 and TRIAC actions were not affected by IOP. These results suggested that BbC/EBPα/βexpression was mainly induced by T3 synthesized through deiodination of T4 in B. belcheri, as in vertebrates.To test if T3, T4 and TRIAC bind to BbTR, a fluorescence spectroscopy was performed. both T3 and TRIAC were able to bind to BbTR, with the TRIAC-BbTR binding being much stronger. In contrast, T4 did not bind to BbTR. At last, we confirm that the endostyle abound in thyroxine (T4) and triiodothyronine (T3), just like the thyroid of vertebrate. And now we know the expression of BbC/EBPα/βis mediated through interaction between T3/TRIAC-TR. Altogether these results suggest the presence in amphioxus of a signaling flow from the endostyle to the hepatic caecum.We have also studied the possible immune role of the yolk protein phosvitin(Pv), derived from vitellogenin of zebrafish. Firstly, we sythesized recombinant Pv, which was identified by MALDI/TOF MS analysis. Then we tested the antibacterial activity of purified Pv and found that Pv have strong antimicrobial activity to both Gram-negative bacterium Escherichia coli (E. coli) and Gram-positive bacterium Staphylococcus aureus (S. aureus). In addition, scanning electron microscope (SEM) showed that both E. coli and S. aureus were damaged by treatment with purified Pv.To test if His-Pv can bind to microbes, FITC-labeled His-Pv was incubated with E. coli and S. aureus. It was found that His-Pv is able to bind both E. coli and S. aureus. To better understand the mechanisms of binding activity, an enzyme-linked immunosorbent assay (ELISA) was carried out to investigate what molecules on the microbial surfaces are recognized by His-Pv, and it was found that His-Pv had a significantly stronger affinity to the immobilized ligands including LPS from Gram-negative bacteria, LTA from Gram-positive bacteria, PGN from both Gram-positive and Gram-negative bacteria than to BSA. These results indicated that His-Pv was a multivalent pattern recognition receptor. Iron binding assay indicated that His-Pv was not able to bind iron, and iron failed to decrease the antimicrobial activity of His-Pv. These reveal that iron ’robbing’is not involved in His-Pv’s bacteriostatic activity. Analysis of the content of Pv at the different periods of embryonic development by ELISA showed that the Pv concentration decreases with development. but until 12 hours, the concentration of Pv within the embryo remains enough for antimicrobial activity. This denoted that at the early stage of embryo development, Pv is possibly a immuodefence player. To search for the "active core" of Pv, two assay were performed, N-terminal truncation assay and point mutation assay. It was found that when N- terminal 194 amino acid residues were removed, the remaining 55 amino acid residues still have antibacterial activity. Preliminary point mutation approach results showed that positively charged amino acid residues of Pv seems to play a key role. In summary, the present study demonstrates that Pv plays an immunedefense role in the zebrafish embryo development, rather than just a nutrients protein. The antibacterial active center of Pv is at the C-terminal, with positively charged amino acid residues and hydrophobic amino acid residues more crucial for the antimicrobial activity.更多还原