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
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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%。
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.
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%。
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.
引文
[1]陈广文,陈晓虹,吕九全.中国淡水三角涡虫属涡虫研究的历史与进展[J].动物学杂志,1993,33(3)：37-41.
[2]Salo E. The power of regeneration and the stem-cell kingdom:freshwater planarians (Platyhelminthes) [J]. BioEssays,2006,28:546-559.
[3]Sa'nchezlvarado A. Freshwater planarian Schmidtamediterranea:embryogenesis, stem-cells and regeneration[J]. Current Opinion in Genetics & Development,2003, 13:438-444.
[4]刘德增.中国的淡水(三肠目)涡虫[J].动物学杂志,1989,24,38-43.
[5]李光鹏.我国涡虫纲分类学的研究[J].动物学杂志,1994,29(2)：58-61.
[6]陈广文.吕九全,马金友.中国淡水三肠目涡虫已知属种及其地理分布[J].动物学报,2001,47：9-12.
[7]李安萍.三角涡虫的生物学研究[J].太原师范专科学校学报,2001,3：23-25.
[8]刘德增.中国淡水涡虫[M],北京：北京师范大学出版社,1993：15-40.
[9]刘德增.我国淡水亚目涡虫分类研究初报.中国动物学会成立60周年纪念论文集[M],北京：中国科学技术出版社,1994,543-548.
[10]李修岭.温度对涡虫再生的影响[J].临汾师范学院学报,2000(6)：321-324.
[11]丁建发.涡虫的习性[J].德师专学报,2004,16(1)：24-26.
[12]李安萍.三角涡虫的生物学研究[J].太原师范专科学校学报,2001,3：201-204.
[13]R.M.亚历克山大.无脊椎动物学：上册[M],北京：北京大学出版社,1985：208-210
[14]Giannakakou P, Gussio R. Acommon pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells [J].Biological Sciences Pharmacology,2000,97(6):2904-2909.
[15]Giannakakou P, Sackett D, Fojo T. Tubulin/Microtubules:Still a Promising Target for New Chemotherapeutic Agents[J]. Journal of the National Cancer Institute, 2000,92(3):182-183.
[16]李建农,蒋建东.微管的生物学特性与药物研究[J].药学学报,2003,38(4)：311-315.
[17]Mandelkow E, Mandelkow EM. Microtubules and microtubule-associated protein[J]. Current opinion in cell Biology,1995,7:72-81.
[18]Oakley CE, Oakley BR. Identification of gamma-tublin, a new member of the tublin superfamily encoded by mipa geneof Aspergillus nidulans[J].Nature,1989, 338 (6217):662-664.
[19]Michiels F, Falkenburg D, Mueller A. M. Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin[J]. Chromosoma,1987,95(6):387-395.
[20]Elizabeth C, Oakley BR, Oakley R. Identification of γ-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans [J]. Nature,1989,338,662-664.
[21]Goldstein LSB, Gunawardena S. Flying through the Drosophilia cytoskeletal genome[J]. Cell Biology,2000,150(2):63-68.
[22]Hideki K, Kimihiko S, Quan GX. Analysis of alpha- and beta-tubulin genes of Bombyx mori using an EST database[J]. Insect Biochemistry and Molecular Biology,2003,33(1):131-137.
[23]Raff EC, Fackenthal JD, Hutchens JA. Microtubule Architecture Specified by a β-Tubulin Isoform[J]. Science,1997,275(5296):70-73.
[24]Rybczynski R, Gilbert LI. Activation of an extracellular signal-ragulated kinases by the insect prothotacicotropic hormone[J]. Molecular and Cellular Endocrino-logy,1998,141(12):141-151.
[25]徐忠东,吴琴.微管蛋白的研究进展[J].安徽教育学院学报,1999,11(2)：73-74.
[26]Amos L. Bending at Microtubule interfaces [J]. Chemistry & Biology,2004,11 (6): 745-747.
[27]秦松柏,攀东,李春杰,许艳丽.小卷蛾斯式线虫β-微管蛋白基因的克隆与序列分析[J].东北林业大学学报,2008,7(36)：72-83
[28]Colombie N, Verollet C, Sampaio P.et al. The Drosophila γ-tubulin small complex subunit Dgrip84 is required for structural and functional integrity of the spindle apparatus[J]. Molecular Biology of the Cell,2006,17 (1):272-282.
[29]Fukushige T, Siddiqui ZK. An alpha-tubulin required for touch sensitivity in C. elegans [J]. Journal of Cell Science,1999,112(3):395-403.
[30]娄慧玲,高巍,周素娟.真核细胞三个微管蛋白新成员的生物学特征[J].生 物学教学,2006,31(12)：4-6
[31]Don W, Cleveland, et al. Number and evolutionary conservation of α-and β-tubulin and cytoplasmicβ-and γ-actin genes β and γ-action genes using specific cloned cDNA probes[J]. Cell,1980,20(1):95-105.
[32]Davud Soifer, Armin H. Enzymatic activity in tubulin preparations:Inreinsic protein kinase activity in lyophilized preparations of tubulin from porcine brain[J]. Biochimica et Biophysica Acta,1972,271(1):182-192.
[33]Eva Nogales, Sharon G, et al. Structure of theαβ tubulin dimer by electron crystallography [J]. Nuture,1998,391:199-203.
[34]王英杰,董萍.微管蛋白参与跨膜信号的传递[J].生命的化学,1994,14(5)：18-19
[35]Kirschner M, Mitchison T. Beyond self-assembly:from microtubules to morphoge-nesis [J].Cell,1986,45(3):329-3421.
[36]Yen TJ, Machlin PS, Cleveland DW, Autoregulated instability of β-tubulin mRNAs by recognition of the nascent amino terminus of β-tubulin[J]. Nature, 1988,334:580-585.
[37]杨勇,王保怀,李芝芬,张有民.微管蛋白的聚合和变性及紫杉醇的影响[J].物理化学学报,1999,2(15)：182-185.
[38]Oakley BR. An abundance of tublins [J].Trends in cell Biology,2000,10(12): 537-542.
[39]Takai Y, Sasaki T, Matozaki T. Small GTP-binding Proteins [J]. Physiological Reviews,2001,81:153-208.
[40]Yang Z, Watson JC. Molecular cloning and characterization of rho, a ras-related small GTP-binding protein from the garden pea[J]. Proceedings of The National Academy of Sciences,1993,90:8732-8736.
[41]Vernoud V, Horton AC, Yang ZB, Nielsen E. Analysis of the small GTPase gene superfamily of Arabidopsis[J]. Plant Physiology,2003,131:1191-1208.
[42]Takai Y, Sasaki T, Matozaki T. Small GTP-binding Proteins[J]. Physiological Reviews,2001,81:153-208.
[43]石峰.植物小G蛋白功能的最新研究进展[J].热带农业科学,2009,29(7)：69-75.
[44]Novick P.et al.Identification of 23 complementat on groups required for post-translational events in the yeast secretory pathay[J]. Cell,1980,21:205-215.
[45]Gallwitz D, et al. A yeast gene encoding a protein homologous to the human c-has/bas proto-oncogene product [J]. Nature,1983,306:704-707.
[46]樊俊蝶,王伟,梁爱华.Rab蛋白的结构、功能及进化[J].生命的化学,2004,24,(1)：33.
[47]OkonenVM, Stenmark H. Role of Rab GTPase in membrane traffic[J]. The Internationl Academy of Cytology,1997, (176):1-85.
[48]Stroup C, Brunger AT. Crystal sytuctures of a rab protein in its inactive and active conformations[J]. Journal of Molecular Biology,2000, (304):585-598.
[49]康巧华,陈巧林,季清洲,任宏伟,茹炳根.G蛋白Rab3acDNA的克隆与表达[J].中国生物化学与分子生物学报,2002,(1)：75-79.
[50]李希,许根兴,王建军,李侠,沈海雁.人Rab31基因克隆和表达[J].中国生物化学与分子生物学报,2000,(5)：653-658.
[51]林慧贤,刘筱斌,李发强,罗文永,刘良式.水稻小GTP蛋白基因Osrba5B基因的克隆和鉴定[J].高技术通讯,2001,(3)：9-14.
[52]Suzanne P, Dikran A. Targeting Rab GTPases to distinct membrancompartmenrs [J].Narure Reviews Molecular Cell Bioliogy,2004,5,886-896.
[53]Dumas J, Zhu J, Connoly JL, Lambright DG. Stureture basis of aetivation and GTP hydrolysis in rab Proteins[J]. Structure,1999, (7):413-423.
[54]ChattoPadhya D, Langsley G, Carson M, Reeacga R, Delueas L, Smith C. Stureture of the nueleotide-binding domain of Palsmodium falcPiarum rab6 in theGDP-bound form[J]. AetaCyrst,2000, (D56):937-944.
[55]Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins [J]. Physiological Reviews,2001,81:153-208.
[56]吕国栋,王俊华,卢晓梅,温浩,林仁勇.细粒棘球绦虫原头蚴和成虫发育调控基因[J].中国寄生虫学与寄生虫病杂志,2009,2(27)：91-93
[57]Stenmark H, Olkkonen VM. The rab GTPase family[J].Genome Biology reviews, 2001,2(5):3007.1-3007.7
[58]Zerial M, McBride H, Rab proteins as membrane organizers [J]. Nature reviews Molecular cell biology,2001,2:107-117.
[59]Marsh M, McMahon HT.The structural Era of Endocytosis[J].Science,1999, 285:215-220.
[60]Rybin V, et al. Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion[J].The EMEBO Journal,1998,17:1930-1940.
[61]Shon C, Farnsworth CL, et al. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis[J]. Narure,1992,359:288-294.
[62]Takai Y, Sasaki T, Matozaki T. Stureture and mutational analysis of Rab GDP--dissoeiation ihnibitor[J]. Narure,1996, (381):42-48.
[63]Janny LP, Filip C, Tom G. For ward genetics and map2 based cloning approaches[J]. Trends in Plant Science,2003,8 (10):484-491.
[64]Kikuch I, Satohk S, Nagat T, et al. Collection mapping and annotation of over 28,000 cDNA clones from Japonica rice[J]. Science,2003,301:376-379.
[65]Nishiyama Y, Motoaki S, Carninci P, et al. High-efficiency cloning of Arabidop sis full-length cDNA by biotinylated CAP trapper[J]. The Plant Journal,1998,15 (5):707-720.
[66]Carninci P, Waki K, Shipaki T, et al. Targeting a complex transcritome:The construction of the mouse full-length cDNA encyclopedia[J]. Genome Research, 2003,13:1273-1289.
[67]Diatchenko L, Lau YC, Campbeu AP,et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries[J]. Proceeding of National Academy of Sciences of the USA,1996,93 (12):6025-6030.
[68]王关林,方宏筠. 植物基因工程(第二版)[M],北京：科学技术出版社,2002：43-170.
[69]Hofstetter H, Schambock A, Van Den Berg J, et al. Sepcific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(A) poly (T) method[J]. Biochimica et Biophysica Ata,1976,454(3):587-591.
[70]宋晓宏,沙伟.cDNA文库技术在植物抗旱机制研究中的应用[J].生物技术通报,2009,3：57-60.
[71]董志敏,张宝石,关荣霞等.全长cDNA文库的构建方法[J].中国农学通报,2006,22(2)：51-55.
[72]谢卡斌,张建伟等.10828条籼稻全长cDNA的分离与注释[J].生命的科学,2005,35(1)：6-12.
[73]毛新国,景蕊莲等.几种全长cDNA文库构建方法比较[J].遗传,2006,28(7)：865-873.
[74]朱利军,长孙东亭等.全长cDNA文库构建方法及应用研究[J].海南大学学报自然科学版,2009,27(2)：185-190
[75]Zhang YH, Qu ZP, Zheng WM, et al. Phylogenetic Analysis of the Sequences of rDNA Internal Transcribed Spacer(ITS) of Phytophthora sojae[J]. Journal of Genrtics and Genomics,2007,34(2):180-188.
[76]Skiba B, Ford R, et al. Construction of a cDNA libraray of Lathyrus sativus with Mycosphaerella pinodes and the expression of potential defence-related expressed sequence tags(ESTa) [J]. Physiological and Molecular Plant Pathology,2005,66: 55-67.
[77]Du LX, Liu SF, Zhu J, et al. Construction of SMART cDNA Library of Sheep Ovary and Identification of Candidate Gene[J]. Agricultural Sciences in China, 2007,6(11):1390-1395.
[78]马金彪,王晓杰等.条锈菌诱导的小麦叶片cDNA文库构建及表达序列标签分析[J].植物病理学报,2007,37(3)：265-270.
[79]Veronika E, Anisimova, et al. Thermolabile duplex-specific nuclease[J]. Biotec-hnology Letters,2009,2(31):251-257.
[80]Zhulidov, Pavel A,et al. Simple cDNA normalization using kamchatka crab dup-lex-specific nuclease[J]. Nucleic Acids Research,2004,32(3):1-8.
[81]张石柱等.利用DSN和SMARTTM技术构建金龟子绿僵菌产孢时期均一化全长cDNA文库[J].农业生物技术学报,2007,15(5)：884-887.
[82]田金良.三角真涡虫生态因子初测[J].动物学杂志,1989,24(4)：40-41.
[83]黄正一,将正撰.物学实验方法[M].上海：上海科学技术出版,1984.
[84]张瑞清.无脊椎动物采集、培养、玻片制作手册[M].北京：北京大学出版社,1988
[85]李强,巫锦雄,易祖盛.涡虫的采集和饲养[J].生物学教学,2009,34(11)：69-70.
[86]周启贵.东亚三角头涡虫的全年采集与培养[J].四川动物,1992,11(3)：44-41.
[87]四川省永川县教师进修学校生地教研组.虫的人工饲养.生物学教学,1990(4)：26-27.
[88]安瑞永,刘敬泽,张路平.水媳和涡虫的立体生态养殖[J].生物学通报,2000,35(1)：35
[89]Sanchez Alvarado A, Kang H. Multicellularity stem cells and the neoblasts of the planarian Schmi dtea medi terranea [J]. Exp Cell Res,2005,306:299-308.
[90]任淑仙.无脊椎动物学[M].上册.北京：北京大学出版社,1990.
[91]郑发科.淡水涡虫的室内培养[J].2008,29(1)：52-55.
[92]吴东,刘俊杰等.中棉所36均一化全长cDNA文库的构建于鉴定[J].作物学报,2009,35(4)：602-607
[93]Yen TJ, Gay DA, Pachter JS, et al. Autoregulated changes in stability of polyribo-some-bound beta-tubulin mRNAs are specified by the first 13 translated nucleti-des[J].Molecular and Cellular Biology,1988,8:1224-1235.
[94]Burns RG, Surridge CD. Functional role of aconsensus peptide which is common to a-,P-,y-tubulin,to action and contraction,to phytochromeA,and to the TCP1α chaperonin protein[J]. FEBS,1994,347(27):105-111.
[95]樊东,秦松柏,朴冬花,许艳丽.二化螟β1微管蛋白基因cDNA序列的克隆与序列分析[J].生物技术通报,2008,4：130-135
[96]王庆浩,陈爱华,张伯礼.丹参：一种中药研究的模式生物[J].中医药学报,2009,37(4)：1-3
[97]王镜岩,朱胜庚,徐长法.生物化学(第三版)[M].北京：高等教育出版社,2004,203
[98]翟中和,王喜忠,丁明孝.细胞生物学[M].北京.高等教育出版社,2000,78-193
[99]Andreas D, Baxevanis BF, Francis Ouellette.生物信息学[M],北京：清华大学出版社,2000：75-80.
[100]Tamura K, Dudley J, Nei M, Kumar S. MAGA4:Molecular Evolutionary Geneti cs Analysis (MEGA) Software Version 4.0[J].Molecular Biology and Evolution, 2007,24:1596-1599.
[101]Saitou N, Nei M. The Neighbor-Joining Method:A New Method for Reconstru-cting Phylogenetic Trees[J].Molecular Biology and Evolution,1987,4:406-425
[102]蔡应繁,谢永芳等.棉花腺体形成时均一化cDNA文库的构建[J].四川大学学报,2007,44(6)：1321-1325
[103]DeRenzis S, Sonnichsen B, Zerial M. Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes[J].Natue Cell Biol- ogy,2002,4:124-133.
[104]Lazar T, et al. Vesicular transport:how many Ypt/Rab-GTPases a eukaryotic cell [J].Trends in biochemical sciences,1997,22:468-472.
[105]Pereira-Leal JB, et al.Evolution of the Rab family of small GTP-binding protein [J].Journal of Molecular Biology,2001,313:889-901.
[106]Ruther ford S, et al.The Arabidopsis Rab-GTPases family:anther enigma varia-ation[J].Current Opinion in plant Biology,2002,5(6):518-528.
[107]Segev N.Ypt and Rab GTPases:insight into function through novel intractions [J]. Current Opinion in Cell Biology,2001,13:500-511
[108]吴文林,戴聪杰,袁建军.日本对虾Rab基因的克隆、结构分析与重组
[109]樊俊蝶,王伟等.游仆虫Rab蛋白基因的克隆表达与纯化[J].中国生物化学与分子生物学报,2004,20(2)：200-205
[110]马克学.淡水涡虫：分类地位、胚胎发生和再生[J].河南师范大学学报,2008.36(4)：140-144.
[111]Zahraoui A. Properties of Rab13 interaction with protein Kinase A[J]. Methods Enzymol,2005,403:723-732.
[112]Sugawara K, et al. Rab11 and its effector Rip11 participate in regulation of insulin granule exocytosis[J]. Genes Cells,2009,14:445-456.
[113]Barbieri MA, et al. Epidermal Growth Factor and Membrane Trafficking Egf Receptor Activation of Endocytosis Requires Rab5a[J].The Journal of Cell Biology,2000,151:539.
[114]杨勇,王保怀,李芝芳,张有民.微管蛋白的聚合和扁形及紫杉醇的影响[J].物理化学学报,1999,15(2)182-185.
[115]Edlind TD, Visvesvara GS. Phylogenetic Analysis of B-Tublin Sequences from Amitochondrial Protozoa[J]. Molecular Phylogenetics and Evolution,1996,2: 359-367.
[2]Salo E. The power of regeneration and the stem-cell kingdom:freshwater planarians (Platyhelminthes) [J]. BioEssays,2006,28:546-559.
[3]Sa'nchezlvarado A. Freshwater planarian Schmidtamediterranea:embryogenesis, stem-cells and regeneration[J]. Current Opinion in Genetics & Development,2003, 13:438-444.
[4]刘德增.中国的淡水(三肠目)涡虫[J].动物学杂志,1989,24,38-43.
[5]李光鹏.我国涡虫纲分类学的研究[J].动物学杂志,1994,29(2)：58-61.
[6]陈广文.吕九全,马金友.中国淡水三肠目涡虫已知属种及其地理分布[J].动物学报,2001,47：9-12.
[7]李安萍.三角涡虫的生物学研究[J].太原师范专科学校学报,2001,3：23-25.
[8]刘德增.中国淡水涡虫[M],北京：北京师范大学出版社,1993：15-40.
[9]刘德增.我国淡水亚目涡虫分类研究初报.中国动物学会成立60周年纪念论文集[M],北京：中国科学技术出版社,1994,543-548.
[10]李修岭.温度对涡虫再生的影响[J].临汾师范学院学报,2000(6)：321-324.
[11]丁建发.涡虫的习性[J].德师专学报,2004,16(1)：24-26.
[12]李安萍.三角涡虫的生物学研究[J].太原师范专科学校学报,2001,3：201-204.
[13]R.M.亚历克山大.无脊椎动物学：上册[M],北京：北京大学出版社,1985：208-210
[14]Giannakakou P, Gussio R. Acommon pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells [J].Biological Sciences Pharmacology,2000,97(6):2904-2909.
[15]Giannakakou P, Sackett D, Fojo T. Tubulin/Microtubules:Still a Promising Target for New Chemotherapeutic Agents[J]. Journal of the National Cancer Institute, 2000,92(3):182-183.
[16]李建农,蒋建东.微管的生物学特性与药物研究[J].药学学报,2003,38(4)：311-315.
[17]Mandelkow E, Mandelkow EM. Microtubules and microtubule-associated protein[J]. Current opinion in cell Biology,1995,7:72-81.
[18]Oakley CE, Oakley BR. Identification of gamma-tublin, a new member of the tublin superfamily encoded by mipa geneof Aspergillus nidulans[J].Nature,1989, 338 (6217):662-664.
[19]Michiels F, Falkenburg D, Mueller A. M. Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin[J]. Chromosoma,1987,95(6):387-395.
[20]Elizabeth C, Oakley BR, Oakley R. Identification of γ-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans [J]. Nature,1989,338,662-664.
[21]Goldstein LSB, Gunawardena S. Flying through the Drosophilia cytoskeletal genome[J]. Cell Biology,2000,150(2):63-68.
[22]Hideki K, Kimihiko S, Quan GX. Analysis of alpha- and beta-tubulin genes of Bombyx mori using an EST database[J]. Insect Biochemistry and Molecular Biology,2003,33(1):131-137.
[23]Raff EC, Fackenthal JD, Hutchens JA. Microtubule Architecture Specified by a β-Tubulin Isoform[J]. Science,1997,275(5296):70-73.
[24]Rybczynski R, Gilbert LI. Activation of an extracellular signal-ragulated kinases by the insect prothotacicotropic hormone[J]. Molecular and Cellular Endocrino-logy,1998,141(12):141-151.
[25]徐忠东,吴琴.微管蛋白的研究进展[J].安徽教育学院学报,1999,11(2)：73-74.
[26]Amos L. Bending at Microtubule interfaces [J]. Chemistry & Biology,2004,11 (6): 745-747.
[27]秦松柏,攀东,李春杰,许艳丽.小卷蛾斯式线虫β-微管蛋白基因的克隆与序列分析[J].东北林业大学学报,2008,7(36)：72-83
[28]Colombie N, Verollet C, Sampaio P.et al. The Drosophila γ-tubulin small complex subunit Dgrip84 is required for structural and functional integrity of the spindle apparatus[J]. Molecular Biology of the Cell,2006,17 (1):272-282.
[29]Fukushige T, Siddiqui ZK. An alpha-tubulin required for touch sensitivity in C. elegans [J]. Journal of Cell Science,1999,112(3):395-403.
[30]娄慧玲,高巍,周素娟.真核细胞三个微管蛋白新成员的生物学特征[J].生 物学教学,2006,31(12)：4-6
[31]Don W, Cleveland, et al. Number and evolutionary conservation of α-and β-tubulin and cytoplasmicβ-and γ-actin genes β and γ-action genes using specific cloned cDNA probes[J]. Cell,1980,20(1):95-105.
[32]Davud Soifer, Armin H. Enzymatic activity in tubulin preparations:Inreinsic protein kinase activity in lyophilized preparations of tubulin from porcine brain[J]. Biochimica et Biophysica Acta,1972,271(1):182-192.
[33]Eva Nogales, Sharon G, et al. Structure of theαβ tubulin dimer by electron crystallography [J]. Nuture,1998,391:199-203.
[34]王英杰,董萍.微管蛋白参与跨膜信号的传递[J].生命的化学,1994,14(5)：18-19
[35]Kirschner M, Mitchison T. Beyond self-assembly:from microtubules to morphoge-nesis [J].Cell,1986,45(3):329-3421.
[36]Yen TJ, Machlin PS, Cleveland DW, Autoregulated instability of β-tubulin mRNAs by recognition of the nascent amino terminus of β-tubulin[J]. Nature, 1988,334:580-585.
[37]杨勇,王保怀,李芝芬,张有民.微管蛋白的聚合和变性及紫杉醇的影响[J].物理化学学报,1999,2(15)：182-185.
[38]Oakley BR. An abundance of tublins [J].Trends in cell Biology,2000,10(12): 537-542.
[39]Takai Y, Sasaki T, Matozaki T. Small GTP-binding Proteins [J]. Physiological Reviews,2001,81:153-208.
[40]Yang Z, Watson JC. Molecular cloning and characterization of rho, a ras-related small GTP-binding protein from the garden pea[J]. Proceedings of The National Academy of Sciences,1993,90:8732-8736.
[41]Vernoud V, Horton AC, Yang ZB, Nielsen E. Analysis of the small GTPase gene superfamily of Arabidopsis[J]. Plant Physiology,2003,131:1191-1208.
[42]Takai Y, Sasaki T, Matozaki T. Small GTP-binding Proteins[J]. Physiological Reviews,2001,81:153-208.
[43]石峰.植物小G蛋白功能的最新研究进展[J].热带农业科学,2009,29(7)：69-75.
[44]Novick P.et al.Identification of 23 complementat on groups required for post-translational events in the yeast secretory pathay[J]. Cell,1980,21:205-215.
[45]Gallwitz D, et al. A yeast gene encoding a protein homologous to the human c-has/bas proto-oncogene product [J]. Nature,1983,306:704-707.
[46]樊俊蝶,王伟,梁爱华.Rab蛋白的结构、功能及进化[J].生命的化学,2004,24,(1)：33.
[47]OkonenVM, Stenmark H. Role of Rab GTPase in membrane traffic[J]. The Internationl Academy of Cytology,1997, (176):1-85.
[48]Stroup C, Brunger AT. Crystal sytuctures of a rab protein in its inactive and active conformations[J]. Journal of Molecular Biology,2000, (304):585-598.
[49]康巧华,陈巧林,季清洲,任宏伟,茹炳根.G蛋白Rab3acDNA的克隆与表达[J].中国生物化学与分子生物学报,2002,(1)：75-79.
[50]李希,许根兴,王建军,李侠,沈海雁.人Rab31基因克隆和表达[J].中国生物化学与分子生物学报,2000,(5)：653-658.
[51]林慧贤,刘筱斌,李发强,罗文永,刘良式.水稻小GTP蛋白基因Osrba5B基因的克隆和鉴定[J].高技术通讯,2001,(3)：9-14.
[52]Suzanne P, Dikran A. Targeting Rab GTPases to distinct membrancompartmenrs [J].Narure Reviews Molecular Cell Bioliogy,2004,5,886-896.
[53]Dumas J, Zhu J, Connoly JL, Lambright DG. Stureture basis of aetivation and GTP hydrolysis in rab Proteins[J]. Structure,1999, (7):413-423.
[54]ChattoPadhya D, Langsley G, Carson M, Reeacga R, Delueas L, Smith C. Stureture of the nueleotide-binding domain of Palsmodium falcPiarum rab6 in theGDP-bound form[J]. AetaCyrst,2000, (D56):937-944.
[55]Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins [J]. Physiological Reviews,2001,81:153-208.
[56]吕国栋,王俊华,卢晓梅,温浩,林仁勇.细粒棘球绦虫原头蚴和成虫发育调控基因[J].中国寄生虫学与寄生虫病杂志,2009,2(27)：91-93
[57]Stenmark H, Olkkonen VM. The rab GTPase family[J].Genome Biology reviews, 2001,2(5):3007.1-3007.7
[58]Zerial M, McBride H, Rab proteins as membrane organizers [J]. Nature reviews Molecular cell biology,2001,2:107-117.
[59]Marsh M, McMahon HT.The structural Era of Endocytosis[J].Science,1999, 285:215-220.
[60]Rybin V, et al. Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion[J].The EMEBO Journal,1998,17:1930-1940.
[61]Shon C, Farnsworth CL, et al. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis[J]. Narure,1992,359:288-294.
[62]Takai Y, Sasaki T, Matozaki T. Stureture and mutational analysis of Rab GDP--dissoeiation ihnibitor[J]. Narure,1996, (381):42-48.
[63]Janny LP, Filip C, Tom G. For ward genetics and map2 based cloning approaches[J]. Trends in Plant Science,2003,8 (10):484-491.
[64]Kikuch I, Satohk S, Nagat T, et al. Collection mapping and annotation of over 28,000 cDNA clones from Japonica rice[J]. Science,2003,301:376-379.
[65]Nishiyama Y, Motoaki S, Carninci P, et al. High-efficiency cloning of Arabidop sis full-length cDNA by biotinylated CAP trapper[J]. The Plant Journal,1998,15 (5):707-720.
[66]Carninci P, Waki K, Shipaki T, et al. Targeting a complex transcritome:The construction of the mouse full-length cDNA encyclopedia[J]. Genome Research, 2003,13:1273-1289.
[67]Diatchenko L, Lau YC, Campbeu AP,et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries[J]. Proceeding of National Academy of Sciences of the USA,1996,93 (12):6025-6030.
[68]王关林,方宏筠. 植物基因工程(第二版)[M],北京：科学技术出版社,2002：43-170.
[69]Hofstetter H, Schambock A, Van Den Berg J, et al. Sepcific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(A) poly (T) method[J]. Biochimica et Biophysica Ata,1976,454(3):587-591.
[70]宋晓宏,沙伟.cDNA文库技术在植物抗旱机制研究中的应用[J].生物技术通报,2009,3：57-60.
[71]董志敏,张宝石,关荣霞等.全长cDNA文库的构建方法[J].中国农学通报,2006,22(2)：51-55.
[72]谢卡斌,张建伟等.10828条籼稻全长cDNA的分离与注释[J].生命的科学,2005,35(1)：6-12.
[73]毛新国,景蕊莲等.几种全长cDNA文库构建方法比较[J].遗传,2006,28(7)：865-873.
[74]朱利军,长孙东亭等.全长cDNA文库构建方法及应用研究[J].海南大学学报自然科学版,2009,27(2)：185-190
[75]Zhang YH, Qu ZP, Zheng WM, et al. Phylogenetic Analysis of the Sequences of rDNA Internal Transcribed Spacer(ITS) of Phytophthora sojae[J]. Journal of Genrtics and Genomics,2007,34(2):180-188.
[76]Skiba B, Ford R, et al. Construction of a cDNA libraray of Lathyrus sativus with Mycosphaerella pinodes and the expression of potential defence-related expressed sequence tags(ESTa) [J]. Physiological and Molecular Plant Pathology,2005,66: 55-67.
[77]Du LX, Liu SF, Zhu J, et al. Construction of SMART cDNA Library of Sheep Ovary and Identification of Candidate Gene[J]. Agricultural Sciences in China, 2007,6(11):1390-1395.
[78]马金彪,王晓杰等.条锈菌诱导的小麦叶片cDNA文库构建及表达序列标签分析[J].植物病理学报,2007,37(3)：265-270.
[79]Veronika E, Anisimova, et al. Thermolabile duplex-specific nuclease[J]. Biotec-hnology Letters,2009,2(31):251-257.
[80]Zhulidov, Pavel A,et al. Simple cDNA normalization using kamchatka crab dup-lex-specific nuclease[J]. Nucleic Acids Research,2004,32(3):1-8.
[81]张石柱等.利用DSN和SMARTTM技术构建金龟子绿僵菌产孢时期均一化全长cDNA文库[J].农业生物技术学报,2007,15(5)：884-887.
[82]田金良.三角真涡虫生态因子初测[J].动物学杂志,1989,24(4)：40-41.
[83]黄正一,将正撰.物学实验方法[M].上海：上海科学技术出版,1984.
[84]张瑞清.无脊椎动物采集、培养、玻片制作手册[M].北京：北京大学出版社,1988
[85]李强,巫锦雄,易祖盛.涡虫的采集和饲养[J].生物学教学,2009,34(11)：69-70.
[86]周启贵.东亚三角头涡虫的全年采集与培养[J].四川动物,1992,11(3)：44-41.
[87]四川省永川县教师进修学校生地教研组.虫的人工饲养.生物学教学,1990(4)：26-27.
[88]安瑞永,刘敬泽,张路平.水媳和涡虫的立体生态养殖[J].生物学通报,2000,35(1)：35
[89]Sanchez Alvarado A, Kang H. Multicellularity stem cells and the neoblasts of the planarian Schmi dtea medi terranea [J]. Exp Cell Res,2005,306:299-308.
[90]任淑仙.无脊椎动物学[M].上册.北京：北京大学出版社,1990.
[91]郑发科.淡水涡虫的室内培养[J].2008,29(1)：52-55.
[92]吴东,刘俊杰等.中棉所36均一化全长cDNA文库的构建于鉴定[J].作物学报,2009,35(4)：602-607
[93]Yen TJ, Gay DA, Pachter JS, et al. Autoregulated changes in stability of polyribo-some-bound beta-tubulin mRNAs are specified by the first 13 translated nucleti-des[J].Molecular and Cellular Biology,1988,8:1224-1235.
[94]Burns RG, Surridge CD. Functional role of aconsensus peptide which is common to a-,P-,y-tubulin,to action and contraction,to phytochromeA,and to the TCP1α chaperonin protein[J]. FEBS,1994,347(27):105-111.
[95]樊东,秦松柏,朴冬花,许艳丽.二化螟β1微管蛋白基因cDNA序列的克隆与序列分析[J].生物技术通报,2008,4：130-135
[96]王庆浩,陈爱华,张伯礼.丹参：一种中药研究的模式生物[J].中医药学报,2009,37(4)：1-3
[97]王镜岩,朱胜庚,徐长法.生物化学(第三版)[M].北京：高等教育出版社,2004,203
[98]翟中和,王喜忠,丁明孝.细胞生物学[M].北京.高等教育出版社,2000,78-193
[99]Andreas D, Baxevanis BF, Francis Ouellette.生物信息学[M],北京：清华大学出版社,2000：75-80.
[100]Tamura K, Dudley J, Nei M, Kumar S. MAGA4:Molecular Evolutionary Geneti cs Analysis (MEGA) Software Version 4.0[J].Molecular Biology and Evolution, 2007,24:1596-1599.
[101]Saitou N, Nei M. The Neighbor-Joining Method:A New Method for Reconstru-cting Phylogenetic Trees[J].Molecular Biology and Evolution,1987,4:406-425
[102]蔡应繁,谢永芳等.棉花腺体形成时均一化cDNA文库的构建[J].四川大学学报,2007,44(6)：1321-1325
[103]DeRenzis S, Sonnichsen B, Zerial M. Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes[J].Natue Cell Biol- ogy,2002,4:124-133.
[104]Lazar T, et al. Vesicular transport:how many Ypt/Rab-GTPases a eukaryotic cell [J].Trends in biochemical sciences,1997,22:468-472.
[105]Pereira-Leal JB, et al.Evolution of the Rab family of small GTP-binding protein [J].Journal of Molecular Biology,2001,313:889-901.
[106]Ruther ford S, et al.The Arabidopsis Rab-GTPases family:anther enigma varia-ation[J].Current Opinion in plant Biology,2002,5(6):518-528.
[107]Segev N.Ypt and Rab GTPases:insight into function through novel intractions [J]. Current Opinion in Cell Biology,2001,13:500-511
[108]吴文林,戴聪杰,袁建军.日本对虾Rab基因的克隆、结构分析与重组
[109]樊俊蝶,王伟等.游仆虫Rab蛋白基因的克隆表达与纯化[J].中国生物化学与分子生物学报,2004,20(2)：200-205
[110]马克学.淡水涡虫：分类地位、胚胎发生和再生[J].河南师范大学学报,2008.36(4)：140-144.
[111]Zahraoui A. Properties of Rab13 interaction with protein Kinase A[J]. Methods Enzymol,2005,403:723-732.
[112]Sugawara K, et al. Rab11 and its effector Rip11 participate in regulation of insulin granule exocytosis[J]. Genes Cells,2009,14:445-456.
[113]Barbieri MA, et al. Epidermal Growth Factor and Membrane Trafficking Egf Receptor Activation of Endocytosis Requires Rab5a[J].The Journal of Cell Biology,2000,151:539.
[114]杨勇,王保怀,李芝芳,张有民.微管蛋白的聚合和扁形及紫杉醇的影响[J].物理化学学报,1999,15(2)182-185.
[115]Edlind TD, Visvesvara GS. Phylogenetic Analysis of B-Tublin Sequences from Amitochondrial Protozoa[J]. Molecular Phylogenetics and Evolution,1996,2: 359-367.