文昌鱼发育相关基因GDF8/11、ACTIVIN和NM23-Bbt2的克隆、表达及分子进化分析

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	文昌鱼发育相关基因GDF8/11、ACTIVIN和NM23-Bbt2的克隆、表达及分子进化分析
其他题名	The cloning, expression and molecular phylogenetic analysis of amphioxus GDF8/11, ACTIVIN and NM23-Bbt2 gene
	邢福国
学位类型	博士
	2007-06-01
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	文昌鱼 Gdf8/11 Activin Nm23-bbt2 分子进化分析
摘要	文昌鱼是头索动物，被认为是现存的与脊椎动物最接近的无脊椎动物，经常被看作是分析从无脊椎动物到脊椎动物进化过程的一种重要的模式动物。在本研究中，我们克隆了青岛文昌鱼的GDF8/11、ACTIVIN和NM23-Bbt2基因，并对这些基因在不同胚胎发育时期和不同成体组织中的表达情况进行了分析，同时分析了这些基因的进化情况。文昌鱼GDF8/11的基因组全长为9.9 kb，包括五个外显子和四个内含子，比其他物种多出两个外显子和两个内含子。在多出的第三个内含子中，我们分离出一个可能的转座因子，这表明这个内含子可能来源于转座子。文昌鱼GDF8/11 cDNA编码一个419个氨基酸的前体多肽，这个前体多肽与软体动物、硬骨鱼类、鸟类和哺乳动物的MSTN以及哺乳动物和斑马鱼的GDF11具有高同源性。系统分析表明文昌鱼GDF8/11位于脊椎动物MSTN和GDF11的根部，这个结果证实MSTN/GDF11来源于同一个祖先基因，并且文昌鱼GDF8/11可能就是他们的共同祖先，产生MSTN和GDF11的基因复制事件发生在脊椎动物分离之前和文昌鱼与脊椎动物分离之后或者分离时。RT-PCR结果表明GDF8/11基因在新受精的细胞、早原肠胚和刀形胚胎中表达，这与哺乳动物中的情况不同。这表明GDF8/11在文昌鱼中除了调节肌肉生长外还可能拥有其他的功能。文昌鱼ACTIVIN的基因组序列长为6.1 kb，启动子大约长为447 bp，其基因组包括两个外显子和一个内含子，外显子/内含子边界严格遵守GT…AG的原则。文昌鱼ACTIVIN基因编码一个410个氨基酸的前体蛋白，前体蛋白包括信号肽、N-末端结构域和C-末端结构域。文昌鱼ACTIVIN基因演绎的氨基酸序列与脊椎动物比较发现ACTIVIN基因比较保守，特别是C-末端生物活性区。系统进化分析表明脊椎动物和无脊椎动物的ACTIVIN基因分别聚在一起，文昌鱼的ACTIVIN则位于脊椎动物ACTIVIN分支的根部，这表明文昌鱼ACTIVIN基因可能是脊椎动物ACTIVIN同源基因的祖先基因。文昌鱼NM23-Bbt2 cDNA包括一个编码171个氨基酸的开放阅读框，序列分析表明文昌鱼NM23-Bbt2与其他物种高度保守，他们都包含高度保守的基元，并且这些基元在NM23的功能中扮演着重要的角色。RT-PCR分析表明文昌鱼NM23-Bbt2在所检测的组织和胚胎发育时期中的非特异性的表达模式。系统分析表明脊椎动物和无脊椎动物NM23-H2分别聚在一起，而文昌鱼NM23-Bbt2位于脊椎动物NM23-H2分支的根部，这表明文昌鱼NM23-Bbt2可能是脊椎动物NM23-H2同源基因的祖先基因。从无脊椎动物到脊椎动物的基因组结构比较表明五个外显子和四个内含子的基因组结构可能产生在文昌鱼与脊椎动物分离之前。
其他摘要	Amphioxus, a cephalochordate, is believed to be the closest living relative of the vertebrate. It is often regarded as an important model animal for phylogenetic studies on the evolution of animals from invertebrate to vertebrate. In this study, GDF8/11, ACTIVIN and NM23-Bbt2 were identified from amphioxus (B. belcheri tsingtausenese), and the expression of these genes in embryo and adult tissues was analyzed by RT-PCR. The evolution of these genes was also analyzed. The full-length genome of the amphioxus GDF8/11 is 9.9 kb. The amphioxus GDF8/11 gene consists of five exons flanked by four introns, which have two more exons and introns than that of other species. In intronIII, a possible transposable element was identified. This suggested this intron might derive from transposon. The amphioxus GDF8/11 cDNA encodes a polypeptide of 419 amino acid residues that showed high homology with MSTNs of molluscan, teleostean, avian, and mammalian species and GDF11 of mammalian and zebrafish species. Phologenetic analysis shows that the GDF8/11 is at the base of vertebrate MSTNs and GDF11s. This result might prove that the GDF8/11 derived from one ancestral gene, and the amphioxus GDF8/11 may be the common ancestral, and also the gene duplication event generating MSTN and GDF11 occurred before the divergence of vertebrates and after or at the divergence of amphioxus from vertebrates. RT-PCR results showed that GDF8/11 gene was expressed in new fertilized cell, early gastrulation and knife-shaped embryo, which was different from that in mammals. It suggested that GDF8/11 gene might possess additional functions other than regulating muscle growth in amphioxus. The genomic sequence of amphioxus ACTIVIN is 6.1 kb long. The promoter was about 447 bp long. Compared with cDNA sequence revealed that there were two exons and one introns and the boundary of exon and intron abided by the role of GT…AG strictly. ACTIVIN gene encoded a 410-amino-acid proprotein. The proprotein contained signal domain, N-terminal domain and C-terminal domain. Comparison of deduced amino acid sequences of amphioxus ACTIVIIN with vertebrate showed that ACTIVIN was conserved. Phylogenetic analysis shows that vertebrate and invertebrate ACTIVIN are each grouped together, with amphioxus falling at the base of vertebrate ACTIVIN clade, and suggests that the amphioxus ACTIVIN may represents the ancestral type of ACTIVIN homologue gene. The NM23-Bbt2 cDNA contains an open reading frame coding for 171 amino acids. Sequence analysis showed that the amphioxus NM23-Bbt2 was highly conserved with that of other species. All of them contained high conserved motifs that play important roles in the function of NM23. RT-PCR reveals a ubiquitous expression pattern of amphioxus NM23-Bbt2 in all adult tissues and it was also expressed in all stages during the embryogenesis. Phylogenetic analysis shows that vertebrate and invertebrate NM23-H2 are each grouped together, with amphioxus falling at the base of vertebrate NM23-H2 clade, and suggests that the amphioxus NM23-Bbt2 may represents the ancestral type of NM23-H2 homologue gene. Comparison of the gene structures (four introns and five exons) of NM23-H2 homologue from invertebrate to vertebrate suggested that the gene structure (four introns and five exons) may be generated before the divergence of amphioxus from vertebrates.
页数	117
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/991
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	邢福国. 文昌鱼发育相关基因GDF8/11、ACTIVIN和NM23-Bbt2的克隆、表达及分子进化分析[D]. 海洋研究所. 中国科学院海洋研究所,2007.

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