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银鲫两个胚胎发育调控相关基因的鉴定及其功能分析
Alternative TitleIdentification and Functional Analysis of Two Genes Related to Embryogenesis in Gibel Carp
尹隽
Subtype博士
Thesis Advisor桂建芳
2007-06-16
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
KeywordCagmdkb基因 Cagapoa-iv基因 表达图式 胚胎发育 银鲫
Abstract在胚胎发育过程中,基因按照特定的时间和空间顺序进行表达并发挥功能。本论文在实验室前期工作的基础上,从已建立的银鲫胚胎原肠期和体节期胚胎差减文库中选取了两个差异表达基因,CagMdkb和CagApoA-IV,对它们在胚胎发育过程中的表达特征和功能进行了研究。 Mdk是一个分泌性的蛋白,在神经发育中有重要的作用,并参与人类肿瘤的形成。在银鲫胚胎发育过程中,CagMdkb基因在原肠期就开始表达,在10体节期时表达量上升到最高并在此之后保持稳定的转录本。Western结果显示胚胎早期有一条19kDa的母源CagMdkb蛋白带。大约在10体节期时,又检测到一条17kDa的蛋白带,推测它可能是剪掉了信号肽的CagMdkb成熟蛋白。在胚胎发育的早期,母源的CagMdkb蛋白能在所有卵裂球的细胞质中被检测到。当胚胎发育到18体节期时,新合成蛋白的信号出现在后脑的一对巨大神经元中。此后,新合成的CagMdkb蛋白延伸到前脑、中脑、后脑的神经元和脊髓的神经纤维中。3A10抗体共定位结果表明这对巨大的神经元是一种中间神经元,即Mauthner神经元。分别在银鲫和斑马鱼的早期胚胎中过量表达CagMdkb基因,都会造成前脑组织的发育受到抑制。注射去除信号肽的mRNA结果提示CagMdkb蛋白作用的发挥还依赖于它的分泌特性。上述结果表明,CagMdkb在对神经系统的早期发育起着重要的作用。同时也说明在鱼类脑的发育过程中,Mdkb基因存在着功能上的保守性。 ApoA-IV是阿朴脂蛋白家族的一个成员。整体原位杂交结果显示早期胚胎中CagApoA-IV的转录本主要分布在卵黄合包层(YSL)上,出苗5天后的幼鱼中CagApoA-IV的转录本仅限于肝脏。在成体组织中,CagApoA-IV的转录本主要集中在肝脏和肠中。胚胎时期新合成的CagApoA-IV蛋白信号最早也出现在卵黄合包层中。在出苗后的幼鱼中,CagApoA-IV蛋白却有着广泛的分布,如脑室区、咽、鳃丝、鳔的内皮细胞、肾小管的上皮复合体、肝血窦、心房和心室、背主动脉、节间血管和胰岛等。当注射反义Morpholino到一胞期胚胎中, CagApoA-IV的蛋白量从原肠期开始减少,而这将导致后期胚胎和幼鱼出现严重的畸形。最明显的畸形表现为脑的萎缩、心血管系统的缺陷和卵黄吸收的障碍。切片分析表明其它的中胚层和内胚层器官也有严重的缺陷,如肝脏、肠、肾脏、鳔等。上述结果表明鱼类阿朴脂蛋白A-IV不仅与消化系统的形成相关,更重要的是它直接影响到脑和心血管系统的发育。CagApoA-IV蛋白量的减少造成脑和心血管系统的缺陷暗示CagApoA-IV基因可能与脑部疾病和心血管类疾病有着很大的相关性。
Other AbstractDuring embryogenesis genes are expressed in temporal and spatial manner to perform functions. Based on our previous work, two differentially expressed genes, CagMdkb and CagApoA-IV, were identified from the suppression subtractive hybridization (SSH) cDNA plasmid libraries between 10-somite embryos and gastrula embryos in gibel carp. Their expression patterns and functions during embryogenesis were investigated in present study. Mdk is a secreted protein that plays an important role in neurogenesis and involves in human tumors. In this study, RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during the following embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein is localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal is detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein is also extended to numerous small neurons in forebrain, midbrain and hindbrain, as well as nerve fibers in spinal cord. Co-localization with 3A10 antibody reveals CagMdkb immunoreactivity on the developing Mauthner neuron, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppresses forebrain structures formation, and CagMdkb function depends on the secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish brain formation. ApoA-IV is a member of apolipoprotein family. Whole-mount in situ hybridization reveals CagApoA-IV is predominantly transcribed at YSL in the early embryos, and later the transcripts are restricted in the liver in 5-day larva. In adult the transcripts are mainly in liver and intestine. The newly synthesized CagApoA-IV protein was also firstly detected in the YSL in early stage. At larvae stage CagApoA-IV protein is abundantly localized in the brain ventricles, in pharynx and gill lamellae, in the endothelium layers of swim bladder, in the epithelial complex of pronephric tubules, in the atria and ventricle of the heart, in the sinusoids of the liver, in the dorsal aorta and intersegmental vessels and in the pancreas islet. When anti-sense morpholinos were injected into 1-cell stage embryos, the expression of the CagApoA-IV was reduced from gastrula stage, which leads to severe deficiencies in embryos and larvae at later stages. The dominant optic deficiencies include the shrink brain, abnormal cardiovascular system and decreased yolk absorbability. The anatomic sections also show severe deficiencies in other mesodermal and endodermal organs such as liver, intestine, kidney, and swim bladder. The data indicate that CagApoA-IV not only plays important roles in digestive system development but also affects brain and cardiovascular system formation which implicates deduced CagApoA-IV expression may related with the diseases in brain and cardiovascular system.
Pages143
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12144
Collection学位论文
Recommended Citation
GB/T 7714
尹隽. 银鲫两个胚胎发育调控相关基因的鉴定及其功能分析[D]. 水生生物研究所. 中国科学院水生生物研究所,2007.
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