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题名	非洲爪蛙分泌型核黄素结合蛋白和叶酸结合在早期胚胎中的表达及功能研究
作者	汤济鑫
学位类别	硕士
答辩日期	2013-05
授予单位	中国科学院研究生院
授予地点	北京
导师	毛炳宇
关键词	核黄素 核黄素结合蛋白 分泌型叶酸结合蛋白 非洲爪蛙 早期胚胎发育 表达图式
其他题名	The expression and function of secreted riboflavin and folate binding proteins in Xenopus early embr
学位专业	细胞生物学
中文摘要	核黄素（维生素B2）和叶酸（维生素B9）同属于B组维生素，在动物代谢与发育过程中起着重要作用。核黄素和叶酸的吸收与转运主要是通过膜结合型的受体或转运蛋白完成的。在低等动物中，存在一类结构同源的分泌性核黄素结合蛋白（Riboflavin binging protein，RfBP）和叶酸结合蛋白（secreted folate binding protein, sFBP），目前对这类分子在胚胎发育中的表达与功能的研究尚不深入。RfBP最早是在鸡蛋的蛋清和蛋黄中发现的，此后在鱼类、两栖类、爬行类中都有序列的报道。 本文克隆了非洲爪蛙（Xenopus laevis）的XRfBP和sFBP，分析其氨基酸序列发现，XRfBP含有18个保守的半胱氨酸，两个与核黄素结合有关的保守位点，两个保守的糖基化位点。XRfBP和sFBP都含有233个氨基酸， N端都含有信号肽序列。在培养细胞中，爪蛙RfBP和sFBP均为分泌性蛋白。特别的，在sFBP 蛋白的C端有一富含碱性氨基酸残基的片段，是Furin蛋白酶的潜在识别位点。通过融合蛋白结构的构建，证实sFBP在分泌过程中其C-末端片段被剪切掉。 原位杂交和RT-PCR检测结果显示，XRfBP是一种母源性表达的基因，30-40期时在胚胎心脏和腹部侧板中胚层部位高量、特异性的表达，提示XRfBP可能在非洲爪蛙胚胎的心脏和腹部内脏器官的发育起到重要作用。sFBP是一种合子型表达的基因，19期特异性的在神经板部位表达，26-29期时在神经管、松果体、粘液腺和泄殖孔部位特异性表达，38-40期时在粘液腺和眼睛部位特异性表达。用特异性Morpholino敲低和过表达方法，下调和过表达XRfBP对爪蛙的早期胚胎发育未产生显著影响。特异性Morpholino敲低sFBP将导致胚胎神经管闭合异常，眼睛变小或缺失，提示sFBP可能参与了神经管和眼睛的发育。上述研究结果为进一步揭示分泌性核黄素结合蛋白和叶酸结合蛋白的功能奠定了基础。
英文摘要	Riboflavin (VB2) and folate (VB9), two members of vitamin B family, play important roles in animal metabolism and embryo development. Riboflavin and folate are absorbed and transported mainly through membrane-associated receptors or transporters. Secreted riboflavin binding protein (RfBP) and secreted folate binding protein (sFBP) have been reported in the lower vertebrates. However, their expression pattern and the function during embryo development are elusive. Riboflavin binding protein (RfBP) was first reported in albumen and yolk of chick egg, and then the homologs were isolated in fish, amphibians and reptiles. Here, we isolated Xenopus laevis riboflavin binding protein (XRfBP) and the secreted folate acid binding protein (sFBP) and studied their expression pattern and function during Xenopus early embryo development. XRfBP and sFBP both encode secreted proteins of 233 amino acids. The conserved eighteen cysteines, two conserved motif responsible for riboflavin binding and the two conserved glycoslated site were also found in XRfBP protein sequence, suggesting its function for riboflavin binding. Interestingly, sFBP contains a Furin protease cleavage site near its C terminal end and the last 23 amino acids were cut off during secrestion. Through in situ hybridization and reverse transcription PCR assays, we found that XfBP is a maternally expressed and is specifically expressed in the heart and lateral plate mesoderm at tailbud stages, suggesting potential roles in heart and ventral mesoderm development. sFBP, is expressed zygotically in the neural plate at neural stages and in the pineal body, cement gland and cloacel opening at early tailbud stages and also the retina of the eye at late tailbud stages. Gain and loss of function experiments were carried out to elucidate their potential function during Xenopus early embryo development. sFBP morphants show delayed neural tube closure at neural stages and small eyes at tailbud stages. Unfortunately, the embryos developed normally whether the RfBP was knocked-down or over-expressed. Collectively, our date may give some clues for the future elucidating the function of the secreted riboflavin and folate binding protein.
语种	中文
公开日期	2013-05-23
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/7442]
专题	昆明动物研究所_发育生物学
推荐引用方式 GB/T 7714	汤济鑫. 非洲爪蛙分泌型核黄素结合蛋白和叶酸结合在早期胚胎中的表达及功能研究[D]. 北京. 中国科学院研究生院. 2013.

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