三角涡虫β微管蛋白和Rab蛋白cDNA文库的克隆及生物信息学分析

【作者】 姚杨；

【导师】 黄原；

【作者基本信息】 陕西师范大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 三角涡虫(Dujesia japonica)是扁形动物门涡虫纲动物,因具有形态学的可塑性及独特的再生能力成为再生生物学、神经生物学和发育生物学研究的模式动物。近年来,涡虫研究进入基因组学和功能基因组学领域,这些研究有助于从基因组水平上揭示后生动物进化、发育和再生的共同机制。本论文应用Clontech公司CreatorTM SMARTTM cDNA Library Construction Kit,构建了涡虫cDNA文库,挑选文库的部分克隆进行测序及序列拼接,获得编码β-微管蛋白和Rab蛋白的全长cDNA序列。β-微管蛋白(β-TUBLIN)是构成微管主要成分的一类高度保守的蛋白,参与微管组装及细胞形态发生、生长和分裂等过程。涡虫β微管蛋白cDNA全长1539 bp,编码444个氨基酸,蛋白质分子量为49672.8,p1为4.82,属亲水性,主要定位于细胞质中,有24个磷酸化位点。结构上含有GTP核苷酸结合位点(GGGTGSG)、N端的转录后调控信号序列(MREI)及β微管蛋白两个保守区域：分别位于氨基酸99-106位(NNWAKGHY)和405-424位(RKAFLHWYTGEGMDEMEFTE), Clustul序列比对表明,涡虫β-微管蛋白与其他8种扁形虫β微管蛋白(日本血吸虫、曼氏裂体吸虫、肝片吸虫、华枝睾吸虫、埃及血吸虫、丝盘虫、细粒棘球绦虫、粒棘球绦虫)相似性为92%-96%,由此可见微管蛋白是生物进化过程中类高度保守的基因。该氨基酸序列在不同物种间有较高的相似性,说明其进化的高度保守性。Rab蛋白是小G蛋白家族成员最多的亚家族,分子量在20-30 KD之间,主要功能表现在物质的跨膜转运和分泌方面。涡虫Rab蛋白cDNA全长2141bp,编码206个氨基酸,蛋白质分子量为23089.3,p1为6.59,属亲水性,主要定位于细胞质中,在氨基酸20、21位之间有信号肽剪切位点。有8个磷酸化位点。含有小G蛋白家族五个保守的鸟苷酸结合区域。涡虫Rab蛋白与其他五种近缘物种的Rab蛋白的相似率为65%-75%。更多还原

【Abstract】 The planarian Dujesia japonica is the representative species of Tricladida in the phylum Platyhelminthes, with body length 10-15 mm and a triangular head, and generally found in nature in the overcast cool fresh water. The morphological plasticity and regenerative capability have made them a good model animal in regeneration biology, neurobiology and developmental biology. In recent years, molecular biology and functional genomics have focused on Planarian. This research will help to reveal the common mechanism about evolution, development and regeneration in metazoan.In order to separate and identify the functional important genes from Dujesia japonica, the total RNA were extracted and used to constructed the cDNA library using SMARTTM cDNA Library Construction Kit. The average inserted cDNA fragments were about 1 kb, and the titer of cDNA library is 6×105cfu/ml.With the help of the bioinformatics to predict the deduced protein’structure and function, we identifiedβ-TUBLIN and RAS protein from the full length cDNA plasmid library.Theβtubulin gene sequence is 1539 bp in length, contains an ORF of 1335 base pairs and encodes a polypeptide of 444 residues with a predicted molecular weight about 49.7kD and pⅠ4.82. There is (3 tubulin signature sequence GGGTGSG and the two conserved amino acid sequence motifs NNWAKGHY at site 99 and RKAFLHWYTGEGMDEMEFTE at site 405 of deduced aminoacid. The analysis of phylogenetic relationship showed that the cloned gene has high amino acid sequences similarity of about 92%-96% with the other knownβtubulin in animals.Rab proteins are monomeric small GTP-binding proteins. The Rab gene sequence is 2141 bp, contains an ORF of 621 base pairs and encodes a polypeptide of 206 residues with a predicted molecular weight about 23.1kD and PI 6.59. Rab protein contain four guanine nucleotide binding domains(GNB), this domain plays a key role in molecular regulation, and are highly conserved among plants, yeasts and animals. The cloned gene has high amino acid sequences similarity of about 65%-75%with the other known Rab proteins in animals.更多还原