【作者】 周丽伟；

【导师】 李庆伟；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2008， 硕士

【摘要】 激肽原是缓激肽的前体,在不同种类的动物中,它们的结构和功能都存在着非常大的差异。在哺乳动物和两栖动物之间,这种差异更加明显。甚至连最保守的缓激肽结构域的合成模式和结构都存在差异。除共有的缓激肽结构域之外,哺乳动物激肽原还具有三个半胱氨酸蛋白酶抑制剂结构域(N末端的一个不含常规的半胱氨酸蛋白酶结合位点,另外两个含常规的半胱氨酸蛋白酶结合位点)。为了更进一步阐明激肽原基因的进化机制,在这篇论文中,利用公共数据库中的EST和基因组数据已识别出19个新的激肽原氨基酸序列(包括哺乳动物、鸟类和鱼类),加上之前经实验证实的激肽原,共收集到了57个激肽原氨基酸序列,同时结合氨基酸序列和基因结构作为分子标记,详细地研究了脊椎动物激肽原基因的系统发育关系。主要的研究结果如下:1)基于邻接法构建的氨基酸序列进化树表明,所有的激肽原被分为两簇,一簇包括哺乳动物、鸟类和鱼类,另一簇仅包括两栖动物。较高的自展验证值支持了该进化树拓扑结构的准确性及预测的激肽原序列的可靠性。2)在这57个激肽原序列中有来自于16个物种的24个激肽原具有明确的内含子-外显子边界,它们被用来进行基因结构的分析。结果表明大部分内含子的位置和相位是保守的,但一些内含子仅存在于哺乳动物和鸟类中,而另一些内含子仅存在于鱼类中。3)对这57个激肽原氨基酸序列除去信号肽之后进行结构保守性的分析,结果表明除缓激肽结构域保守的存在于所有物种中之外,其它一些结构域的存在则具有种属特异性。4)对两栖动物缓激肽结构域的进一步分析还发现,两栖动物皮肤分泌物对于特异捕食者的防御机制是缓激肽受体依赖的。5)为了更清晰地揭示脊椎动物激肽原基因的进化,在这个研究中,从现存最古老的无颌类脊椎动物七鳃鳗(Lampetra japonica)肝脏中克隆出了激肽原基因,其开放阅读框含546个核苷酸,编码181个氨基酸残基。对氨基酸序列的分析表明,其具有一个无常规半胱氨酸蛋白酶结合位点的半胱氨酸蛋白酶抑制剂结构域和一个氨基酸残基发生了部分替换的缓激肽结构域。6)利用RT-PCR技术,检测出除了肝脏外,激肽原也在七鳃鳗的肠、肾和白细胞等组织中表达,但不存在于口腔腺中。综合上述的研究结果得出:在现存最原始的脊椎动物七鳃鳗中,激肽原基因编码一个缓激肽结构域和一个半胱氨酸蛋白酶抑制剂结构域。随着物种的进化,该基因逐步加入了其它一些序列,其所编码的蛋白质也逐渐多功能化。特别地是,在两栖动物中,激肽原基因特化出一种仅编码单一缓激肽结构域的类型,其在不种的两栖动物中频繁地发生着多倍化,而非特化的常规类型的激肽原基因则成为假基因,并且在序列上发生了极大的退化,但其基因结构仍然保守。本文的结果为更深入地揭示激肽原以及其它基因家族的进化机制提供了理论基础。更多还原

【Abstract】 Kininogens, the precursors of bradykinins, vary extremely in both structure and function among different taxa of animals, in particular between mammals and amphibians. This includes even the most conserved bradykinin domain in terms of biosynthesis mode and structure. Besides the bradykinin domain, mammalian kininogen also contains three cystatin domains (the one in N-terminal part without canonical cysteine protease binding sites; the other two with those). To further elucidate the evolutionary dynamics of kininogen genes, in this work, we have identified 19 novel amino acid sequences from EST and genomic databases (for mammals, birds, and fishes), and explored their phylogenetic relationships using combined amino acid sequence and gene structure as markers.These mainly results were as follow:1) The topology of the neighbor-joining tree presented that all 57 kininogen amino acid sequences were classified into two clusters, one including mammals, birds, and fishes and the other including only amphibians. The high bootstrap values supported both the precision of topology and the reliability of predicted sequences.2) A total of 24 kininogen sequences from 16 species with clear intron–exon boundaries were used for gene structure analyses. Results suggested strong conservation in terms of both position and phase of most introns, though some introns were found only in certain mammals and birds or fishes.3) The conservative analyses of 57 kininogen amino acid sequences without signal peptides showed that all of the sequences shared the bradykinin domain, whereas other domains were conserved more or less in some taxa of animals.4) We also propose that the defense mechanism against specific predators in amphibian skin secretions has been bradykinin receptor dependent according to the detailed analysis of amphibian bradykinin domains.5) In addition, we also cloned a kininogen gene, which contained a 546bp open reading frame, and encoded 181 amino acids residues, from the liver of lamprey, Lampetra japonica, which is one of the most primitive jawless vertebrates still living today. Protein sequence analyses showed that there were a cystatin domain without canonical cysteine protease binding sites and a bradykinin domain with substitutions of amino acid residues.6) Using reverse transcription PCR, the kininogen mRNA was also detected in lamprey gut, kidney, and leukocyte, but absent in lamprey buccal gland. These above results seem to demonstrate that in lampreys, a kininogen gene encoded a cystatin domain and a bradykinin domain. During the process of vertebrate evolution, the gene was fused by some sequences and encoded proteins with more functions, gradually. Particularly, in amphibians, there was a special kininogen gene, only encoding the bradykinin domain, however, with multiple copies in different species. The canonical kininogen gene became a pseudogene and largely degenerated, but with gene structure conserved.Our findings may provide a foundation for identification and structural, functional, and evolutionary analyses of more kininogen genes and other gene families.更多还原