蒋玉祥本科毕业论文

分类号—————————————————————————————————密级—(宋体小五号)UDC本科毕业论文七鳃鳗（Petromyzonmarinus）类胰岛素生长因子结合蛋白3的基因结构和功能研究学生姓名蒋玉祥学号040012006029指导教师周建峰教授院、系、中心海洋生命学院专业年级06生物化学与分子生物学论文答辩日期年月日中国海洋大学七鳃鳗（Petromyzonmarinus）类胰岛素生长因子结合蛋白3的基因结构和功能研究完成日期：指导教师签字：答辩小组成员签字：七鳃鳗（Petromyzonmarinus）类胰岛素生长因子结合蛋白3的基因结构和功能研究i七鳃鳗（Petromyzonmarinus）类胰岛素生长因子结合蛋白3的基因结构和功能研究摘要IGF结合蛋白（IGFBP）-3是一种细胞外分泌蛋白，它能结合并调控IGF信号。IGFBP-3在培养的哺乳动物细胞中还会表现出配体非依赖的功能。除了IGF结合域（IBD），哺乳动物IGFBP-3还有一个核定位信号（NLS）和转录激活域（TA）。但是，IGFBP-3的这些结构域的体内功能尚不明确。本研究显示在无颌类七鳃鳗IGFBP-3中这些结构域在进化上也是保守的，其中IGFBP-3ORF1含IGF结合结构域、核定位信号和转录激活结构域，分别可以与IGF结合、能定位于细胞核，有转录激活活性。通过构建数个IGFBP-3ORF1突变体来研究其功能，显微注射IGFBP-3ORF1和突变体的mRNA可使斑马鱼的胚胎背部化，发现这种背部化作用是IGF非依赖性的，并且不依赖于核定位信号和转录激活结构域。关键词：七鳃鳗IGFIGFBP-3突变体中国海洋大学学士毕业论文iiGenestructureandfunctionstudyofIGFBP3insealamprey(Petromyzonmarinus)AbstractIGFbindingprotein(IGFBP)-3isasecretedproteinthatbindsandmodulatesIGFactionsinextracellularenvironments.IGFBP-3alsohasligand-independentactionsinculturedmammaliancells.InadditiontoitsIGFbindingdomain(IBD),mammalianIGFBP-3hasafunctionalnuclearlocalizationsignal(NLS)andtransactivationdomain(TA).ThespecificrolesofthesestructuraldomainsofIGFBP-3inmediatingitsinvivoactionsareunclear.ThestudyshowsinsealampreythedomainsofIGFBP3areevolutionarilyconserved.Tostudythefunctionalrolesofthesedomains,severalIGFBP-3ORF1mutantswereengineered.TheIBDmutantfailedtobindIGF-Ibuthadnormalnuclearlocalization.TheNLSmutanthadgreatlyreducednuclearlocalizationbutshowednormalIGFbinding.TheIBD+NLSdoublemutanthadimpairedIGFbindingandnuclearlocalization.Whenexpressedinzebrafishembryos,wild-typeIGFBP-3,NLS,andTAmutantshadstrongdorsalizingeffects.TheactivitiesoftheIBDmutantandtheIBD+NLSmutantweresignificantlylower,suggestingthatIGFBP-3actsinIGF-dependentand-independentmannersandthattheIGF-independentactionisnotrelatedtoitsnuclearlocalizationortransactivationactivity.Keywords:sealampreyIGFIGFBP3mutant中国海洋大学学士毕业论文七鳃鳗（Petromyzonmarinus）类胰岛素生长因子结合蛋白3的基因结构和功能研究III目录1文献综述 8IGF信号通路系统 81.1.1IGF信号通路系统概述 81.1.2IGFBP-3概述 91.2七鳃鳗与基因倍增 111.2.1七鳃鳗概述 111.2.2基因倍增 111.3本研究的目的和意义 132材料和方法 142.1化学试剂 142.2实验仪器 152.3实验材料 152.4Blast分析和序列比对 152.5IGFBP-3的系统进化分析 152.6突变体质粒构建 162.7细胞培养和转染 162.8Western免疫印迹 172.9配体印迹 182.10统计分析 193实验结果 193.1七鳃鳗IGFBP-3基因的序列分析 193.2七鳃鳗IGFBP-3具有保守的且有功能的IGF结合结构域，核定位信号和转录激活结构域 223.3七鳃鳗IGFBP-3可以使斑马鱼胚胎背部化 243.4七鳃鳗IGFBP-3对斑马鱼胚胎的背部化作用不依赖于IGF结合结构域，核定位信号和转录激活结构域 243.5IGFBP-3的功能在进化上是保守的 264讨论与展望 27参考文献 30附录 36附录1主要试剂配方 36附录2用于进化分析和序列比对的蛋白序列信息 37致谢 39中国海洋大学学士毕业论文七鳃鳗（Petromyzonmarinus）类胰岛素生长因子结合蛋白3的基因结构和功能研究PAGE40 PAGE391文献综述IGF信号通路系统1.1.1IGF信号通路系统概述生物体的生长、发育、生理状况以及疾病的发生和发展受体内多种信号通路的调控，主要有BMP信号通路、Wnt信号通路、TGF-β信号通路、Nodal信号通路、Notch信号通路、FGF信号通路、Hedgehog信号通路和IGF信号通路。其中，IGF（Insulin-likegrowthfactor）信号通路是一个在进化上相对保守的信号通路，该信号通路由两个IGF配体（IGF-1和-2）、两个IGF受体（IGF-1R和-2R）和6个IGF结合蛋白（IGF-bindingprotein1-6,IGFBP1-6）组成（图1.1）。研究表明IGF信号系统在调节细胞的增殖、生长、分化、凋亡和迁移等方面发挥其重要的生物学作用，以及在生物体内调节胚胎的生长、发育、维持成体稳态和许多疾病的发生、发展过程中发挥关键作用ADDINEN.CITEADDINEN.CITE.DATA[1-3]。图1.1IGF信号途径的组分和作用模式示意图。IGF信号系统包括两种配体（IGF-1和IGF-2），两种受体（IGF-1R和IGF-2R）和六种IGF结合蛋白（IGFBP-1~6）。在血液循环中，大部分IGF与相应的IGFBP结合，而IGFBP-3和IGFBP-5能与IGF和酸不稳定亚基（ALS）结合形成三元复合物。IGF通过结合相应受体，激活独特的信号通路来发挥作用。1.1.2IGFBP-3概述IGFBP-3是血液中最丰富的IGFBPADDINEN.CITE<EndNote><Cite><Author>Firth</Author><Year>2002</Year><RecNum>6</RecNum><record><rec-number>6</rec-number><foreign-keys><keyapp="EN"db-id="zf5r9dwwdfp5p2ef0xk5r2t7dax5tv0e9dsr">6</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Firth,S.M.</author><author>Baxter,R.C.</author></authors></contributors><auth-address>KollingInstituteofMedicalResearch,UniversityofSydney,RoyalNorthShoreHospital,StLeonards,NewSouthWales2065,Australia.</auth-address><titles><title>Cellularactionsoftheinsulin-likegrowthfactorbindingproteins</title><secondary-title>EndocrRev</secondary-title></titles><periodical><full-title>EndocrRev</full-title></periodical><pages>824-54</pages><volume>23</volume><number>6</number><edition>2002/12/06</edition><keywords><keyword>AminoAcidSequence</keyword><keyword>Animals</keyword><keyword>CellDivision/physiology</keyword><keyword>CellMovement/physiology</keyword><keyword>Humans</keyword><keyword>Insulin-LikeGrowthFactorBindingProteins/*physiology</keyword><keyword>Mice</keyword><keyword>Mice,Knockout</keyword><keyword>Mice,Transgenic</keyword><keyword>MolecularSequenceData</keyword><keyword>Receptors,Somatomedin/physiology</keyword><keyword>SignalTransduction/physiology</keyword><keyword>Somatomedins/physiology</keyword></keywords><dates><year>2002</year><pub-dates><date>Dec</date></pub-dates></dates><isbn>0163-769X(Print)</isbn><accession-num>12466191</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12466191</url></related-urls></urls><language>eng</language></record></Cite></EndNote>[3]。它与IGF以及酸不稳定亚基（acid-labilesubunit,ALS）形成三重复合体，保护IGF不被降解，延长IGF的半衰期，并且调节IGF在组织的生物利用度ADDINEN.CITEADDINEN.CITE.DATA[3-5]。另外，这个复合体还可以防止由于血液里高浓度IGF引起的低血糖症。IGFBP-3具有IGF信赖性（IGF-dependent）作用，依赖于不同的细胞系，实验条件等的不同，表现为促进或抑制IGF的作用ADDINEN.CITEADDINEN.CITE.DATA[6]。越来越多的证据显示IGFBP-3具有IGF非信赖性（IGF-independent）作用ADDINEN.CITEADDINEN.CITE.DATA[3,5,7]，并且可以定位于细胞核ADDINEN.CITEADDINEN.CITE.DATA[3,7-9]而与视黄醛受体α（RXR-α）结合ADDINEN.CITEADDINEN.CITE.DATA[10-12]。而且，哺乳动物IGFBP-3的N结构域含有一个转录激活域ADDINEN.CITE<EndNote><Cite><Author>Zhao</Author><Year>2006</Year><RecNum>229</RecNum><record><rec-number>229</rec-number><foreign-keys><keyapp="EN"db-id="zf5r9dwwdfp5p2ef0xk5r2t7dax5tv0e9dsr">229</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Zhao,Y.</author><author>Yin,P.</author><author>Bach,L.A.</author><author>Duan,C.</author></authors></contributors><auth-address>DepartmentofMolecular,Cellular,andDevelopmentalBiology,UniversityofMichigan,AnnArbor,Michigan48109,USA.</auth-address><titles><title>SeveralacidicaminoacidsintheN-domainofinsulin-likegrowthfactor-bindingprotein-5areimportantforitstransactivationactivity</title><secondary-title>JBiolChem</secondary-title></titles><pages>14184-91</pages><volume>281</volume><number>20</number><edition>2006/03/18</edition><keywords><keyword>AminoAcidSequence</keyword><keyword>CellLine</keyword><keyword>Histones/chemistry</keyword><keyword>Humans</keyword><keyword>Insulin-LikeGrowthFactorBindingProtein2/chemistry</keyword><keyword>Insulin-LikeGrowthFactorBindingProtein3/chemistry</keyword><keyword>Insulin-LikeGrowthFactorBindingProtein5/*chemistry/metabolism</keyword><keyword>MolecularSequenceData</keyword><keyword>Mutation</keyword><keyword>ProteinStructure,Tertiary</keyword><keyword>SequenceHomology,AminoAcid</keyword><keyword>Transcription,Genetic</keyword><keyword>*TranscriptionalActivation</keyword></keywords><dates><year>2006</year><pub-dates><date>May19</date></pub-dates></dates><isbn>0021-9258(Print) 0021-9258(Linking)</isbn><accession-num>16543235</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16543235</url></related-urls></urls><electronic-resource-num>M506941200[pii] 10.1074/jbc.M506941200</electronic-resource-num><language>eng</language></record></Cite></EndNote>[13]。最近研究发现IGFBP-3具有IGF非依赖性的作用（Zhongetal.,submitted）。跟其他IGFBP一样，IGFBP-3由信号肽及三个大小相近（~80残基）的结构域组成：N-末-端结构域、C-端结构域和可变的L连接区域组成（图1.2）ADDINEN.CITEADDINEN.CITE.DATA[3,5]。N-端结构域包括12个保守的半胱氨酸残基和主要的与IGF结合的位点（氨基酸56~81）ADDINEN.CITEADDINEN.CITE.DATA[14-17]。C-端结构域包括6个保守的半胱氨酸残基，能介导IGFBP-3和其它蛋白分子相互作用，如IGFBP-3可以通过C-端结构域和酸不稳定性亚单位（Acid-lablesubunit,ALS）ADDINEN.CITEADDINEN.CITE.DATA[3,5]，另外C末端区域对IGF的结合也有一定作用ADDINEN.CITE<EndNote><Cite><Author>Clemmons</Author><Year>2001</Year><RecNum>3152</RecNum><record><rec-number>3152</rec-number><foreign-keys><keyapp="EN"db-id="taraa50ehz5ptce0pt9pz0to2x20fpdzdfd9">3152</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Clemmons,D.R.</author></authors></contributors><auth-address>DepartmentofMedicine,UniversityofNorthCarolina,ChapelHill,NorthCarolina27599-7170,USA.endo@</auth-address><titles><title>UseofmutagenesistoprobeIGF-bindingproteinstructure/functionrelationships</title><secondary-title>EndocrRev</secondary-title></titles><periodical><full-title>EndocrRev</full-title></periodical><pages>800-17</pages><volume>22</volume><number>6</number><edition>2001/12/12</edition><keywords><keyword>AminoAcidSequence</keyword><keyword>BindingSites</keyword><keyword>CarrierProteins/metabolism</keyword><keyword>Endopeptidases/metabolism</keyword><keyword>ExtracellularMatrix/metabolism</keyword><keyword>Glycoproteins/metabolism</keyword><keyword>Glycosaminoglycans/metabolism</keyword><keyword>Humans</keyword><keyword>Insulin-LikeGrowthFactorBinding</keyword><keyword>Proteins/*chemistry/*genetics/physiology</keyword><keyword>Insulin-LikeGrowthFactorI/metabolism/pharmacology</keyword><keyword>Insulin-LikeGrowthFactorII/metabolism/pharmacology</keyword><keyword>Models,Molecular</keyword><keyword>MolecularSequenceData</keyword><keyword>MolecularStructure</keyword><keyword>*Mutagenesis</keyword><keyword>Structure-ActivityRelationship</keyword></keywords><dates><year>2001</year><pub-dates><date>Dec</date></pub-dates></dates><isbn>0163-769X(Print) 0163-769X(Linking)</isbn><accession-num>11739334</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11739334</url></related-urls></urls><language>eng</language></record></Cite></EndNote>[15]。N-端结构域和C-端结构域之间的半胱氨酸能形成区域间二硫桥来稳定整体结构ADDINEN.CITEADDINEN.CITE.DATA[18,19]。中间的L连接区域在IGFBPs之间差异较大，但是连接区域内部包括一些转录后调控位点，包括糖基化、磷酸化和蛋白质水解位点ADDINEN.CITEADDINEN.CITE.DATA[3,5]。图1.2人IGFBP-3的结构，引自ADDINEN.CITE<EndNote><Cite><Author>Jogie-Brahim</Author><Year>2009</Year><RecNum>337</RecNum><record><rec-number>337</rec-number><foreign-keys><keyapp="EN"db-id="zf5r9dwwdfp5p2ef0xk5r2t7dax5tv0e9dsr">337</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Jogie-Brahim,S.</author><author>Feldman,D.</author><author>Oh,Y.</author></authors></contributors><auth-address>DepartmentofPathology,MedicalCollegeofVirginiaCampus,VirginiaCommonwealthUniversity,Richmond,Virginia23298-0662,USA.</auth-address><titles><title>Unravelinginsulin-likegrowthfactorbindingprotein-3actionsinhumandisease</title><secondary-title>EndocrRev</secondary-title></titles><periodical><full-title>EndocrRev</full-title></periodical><pages>417-37</pages><volume>30</volume><number>5</number><edition>2009/05/30</edition><keywords><keyword>ActiveTransport,CellNucleus</keyword><keyword>Animals</keyword><keyword>CellDifferentiation</keyword><keyword>CellNucleus/metabolism</keyword><keyword>CellProliferation</keyword><keyword>ConservedSequence</keyword><keyword>GeneExpression</keyword><keyword>Humans</keyword><keyword>Insulin-LikeGrowthFactorBindingProteins/chemistry/genetics/*physiology</keyword><keyword>Neoplasms/*epidemiology</keyword><keyword>Polymorphism,Genetic</keyword><keyword>ProteinBinding</keyword><keyword>Receptor,IGFType1/metabolism</keyword><keyword>Receptors,CellSurface</keyword><keyword>RiskFactors</keyword><keyword>Somatomedins/*metabolism</keyword></keywords><dates><year>2009</year><pub-dates><date>Aug</date></pub-dates></dates><isbn>1945-7189(Electronic) 0163-769X(Linking)</isbn><accession-num>19477944</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19477944</url></related-urls></urls><electronic-resource-num>er.2008-0028[pii] 10.1210/er.2008-0028</electronic-resource-num><language>eng</language></record></Cite></EndNote>[7]。图中标明了IGFBP-3的三个结构域，并列出了这些主要功能域中的重要功能及结构基序。图中竖线表示半胱氨酸。此外，IGFBP-3的N-端结构域还有转录激活活性结构域（Tansactivationactivitydoman，TA），具有转录激活活性ADDINEN.CITE<EndNote><Cite><Author>Zhao</Author><Year>2006</Year><RecNum>3163</RecNum><record><rec-number>3163</rec-number><foreign-keys><keyapp="EN"db-id="taraa50ehz5ptce0pt9pz0to2x20fpdzdfd9">3163</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Zhao,Y.</author><author>Yin,P.</author><author>Bach,L.A.</author><author>Duan,C.</author></authors></contributors><auth-address>DepartmentofMolecular,Cellular,andDevelopmentalBiology,UniversityofMichigan,AnnArbor,Michigan48109,USA.</auth-address><titles><title>SeveralacidicaminoacidsintheN-domainofinsulin-likegrowthfactor-bindingprotein-5areimportantforitstransactivationactivity</title><secondary-title>JBiolChem</secondary-title></titles><periodical><full-title>JBiolChem</full-title></periodical><pages>14184-91</pages><volume>281</volume><number>20</number><edition>2006/03/18</edition><keywords><keyword>AminoAcidSequence</keyword><keyword>CellLine</keyword><keyword>Histones/chemistry</keyword><keyword>Humans</keyword><keyword>Insulin-LikeGrowthFactorBindingProtein2/chemistry</keyword><keyword>Insulin-LikeGrowthFactorBindingProtein3/chemistry</keyword><keyword>Insulin-LikeGrowthFactorBindingProtein5/*chemistry/metabolism</keyword><keyword>MolecularSequenceData</keyword><keyword>Mutation</keyword><keyword>ProteinStructure,Tertiary</keyword><keyword>SequenceHomology,AminoAcid</keyword><keyword>Transcription,Genetic</keyword><keyword>*TranscriptionalActivation</keyword></keywords><dates><year>2006</year><pub-dates><date>May19</date></pub-dates></dates><isbn>0021-9258(Print) 0021-9258(Linking)</isbn><accession-num>16543235</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16543235</url></related-urls></urls><electronic-resource-num>M506941200[pii] 10.1074/jbc.M506941200</electronic-resource-num><language>eng</language></record></Cite></EndNote>[13]。C-端结构域还具有核定位信号序列（Nuclearlocalizationsequence,NLS），位于氨基酸215~232之间ADDINEN.CITEADDINEN.CITE.DATA[3,8,9,20]，其中228KGRKR232为5个关键氨基酸，将这5个氨基酸突变为相应于人的IGFBP-1的序列MDEGA时可以完全破坏它的入核活性ADDINEN.CITEADDINEN.CITE.DATA[20-22]；另外，在C-端结构域还有肝素结合基序（Heparinbindingmotif,HBM），细胞表面的粘多糖（Glycosaminoglycan,GAG）侧链以及细胞外的基质（Extracelluarmatrix,ECM）蛋白结合ADDINEN.CITE<EndNote><Cite><Author>Firth</Author><Year>2002</Year><RecNum>6</RecNum><record><rec-number>6</rec-number><foreign-keys><keyapp="EN"db-id="zf5r9dwwdfp5p2ef0xk5r2t7dax5tv0e9dsr">6</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Firth,S.M.</author><author>Baxter,R.C.</author></authors></contributors><auth-address>KollingInstituteofMedicalResearch,UniversityofSydney,RoyalNorthShoreHospital,StLeonards,NewSouthWales2065,Australia.</auth-address><titles><title>Cellularactionsoftheinsulin-likegrowthfactorbindingproteins</title><secondary-title>EndocrRev</secondary-title></titles><periodical><full-title>EndocrRev</full-title></periodical><pages>824-54</pages><volume>23</volume><number>6</number><edition>2002/12/06</edition><keywords><keyword>AminoAcidSequence</keyword><keyword>Animals</keyword><keyword>CellDivision/physiology</keyword><keyword>CellMovement/physiology</keyword><keyword>Humans</keyword><keyword>Insulin-LikeGrowthFactorBindingProteins/*physiology</keyword><keyword>Mice</keyword><keyword>Mice,Knockout</keyword><keyword>Mice,Transgenic</keyword><keyword>MolecularSequenceData</keyword><keyword>Receptors,Somatomedin/physiology</keyword><keyword>SignalTransduction/physiology</keyword><keyword>Somatomedins/physiology</keyword></keywords><dates><year>2002</year><pub-dates><date>Dec</date></pub-dates></dates><isbn>0163-769X(Print)</isbn><accession-num>12466191</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12466191</url></related-urls></urls><language>eng</language></record></Cite></EndNote>[3]；C-端结构域还有一个能与转铁蛋白（Transferrin）或小窝蛋白（Caveolin）结合的结构域（234FCWCVDKY241）ADDINEN.CITEADDINEN.CITE.DATA[23]ADDINEN.CITEADDINEN.CITE.DATA
，以及金属结合结构域（Metal-bindingdomain）（223QCRPSKGRKRGFCW236）ADDINEN.CITEADDINEN.CITE.DATA[24]。1.2七鳃鳗与基因倍增1.2.1七鳃鳗概述七鳃鳗与盲鳗同属圆口纲（Cyclostomata），是现存脊椎动物中最原始的一纲。它们无上、下颌，因此又称无颌类（Agnatha），都有一个圆形的口吸盘，故又称圆口类。它们栖居于海水或淡水中，营寄生或半寄生生活。无成对的附肢（脊椎动物门中唯一没有附肢的动物），终生保留脊索，没有真正的脊椎骨，只有一些软骨小弧片直立于脊索上方及神经管的两侧，是脊椎的雏形（脊椎动物笫一阶段进化的代表特征）。具有独特的呼吸器官─鳃囊，囊壁为由内胚层来源的褶皱状鳃丝，遍布大量的微血管，可供气体交换。进化地位上它们处在1R之后，2R之前，因此它们是研究脊椎动物功能进化的极好材料。1.2.2基因倍增OhnoADDINEN.CITE<EndNote><Cite><Author>Ohno</Author><Year>1970</Year><RecNum>310</RecNum><record><rec-number>310</rec-number><foreign-keys><keyapp="EN"db-id="zf5r9dwwdfp5p2ef0xk5r2t7dax5tv0e9dsr">310</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Ohno,S.</author></authors></contributors><titles><title>EvolutionbyGeneDuplication</title><secondary-title>Springer-Verlag,Heidelberg</secondary-title></titles><dates><year>1970</year></dates><urls></urls></record></Cite></EndNote>[25]认为，基因，特别是基因组的倍增意义重大，因为可以在短时间内产生大量的遗传物质从而可以用于基因突变和正向筛选产生具有新功能的基因。因为头索动物文昌鱼的基因组大小是尾索动物海鞘的三倍，Ohno认为在尾索动物分化后有过一次基因倍增。对文昌鱼和海鞘的同工酶和同源基因分析，表明大多数基因是单拷贝的，而无颌类的基因组，如七鳃鳗和盲鳗的，包含至少两个直系同源基因，哺乳类包含三个甚至更多ADDINEN.CITE<EndNote><Cite><Author>Holland</Author><Year>1994</Year><RecNum>3410</RecNum><record><rec-number>3410</rec-number><foreign-keys><keyapp="EN"db-id="taraa50ehz5ptce0pt9pz0to2x20fpdzdfd9">3410</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Holland,P.W.</author><author>Garcia-Fernandez,J.</author><author>Williams,N.A.</author><author>Sidow,A.</author></authors></contributors><auth-address>DepartmentofZoology,UniversityofOxford,UK.</auth-address><titles><title>Geneduplicationsandtheoriginsofvertebratedevelopment</title><secondary-title>DevSuppl</secondary-title></titles><periodical><full-title>DevSuppl</full-title></periodical><pages>125-33</pages><edition>1994/01/01</edition><keywords><keyword>AminoAcidSequence</keyword><keyword>Animals</keyword><keyword>BaseSequence</keyword><keyword>Chordata,Nonvertebrate/genetics</keyword><keyword>*Evolution</keyword><keyword>GeneExpression</keyword><keyword>Genes,Homeobox</keyword><keyword>Genome</keyword><keyword>Insulin/genetics</keyword><keyword>MolecularSequenceData</keyword><keyword>*MultigeneFamily</keyword><keyword>Phylogeny</keyword><keyword>Proto-Oncogenes</keyword><keyword>SequenceHomology</keyword><keyword>Urochordata/genetics</keyword><keyword>Vertebrates/*embryology/genetics</keyword></keywords><dates><year>1994</year></dates><accession-num>7579513</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7579513</url></related-urls></urls><language>eng</language></record></Cite></EndNote>[26]。另外文昌鱼中仅有一个Hox基因簇ADDINEN.CITE<EndNote><Cite><Author>Holland</Author><Year>1994</Year><RecNum>154</RecNum><record><rec-number>154</rec-number><foreign-keys><keyapp="EN"db-id="xfx2zrs5b2wt5besdpwxts9lv2szdfd5vpsp">154</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Holland,P.W.</author><author>Garcia-Fernandez,J.</author><author>Williams,N.A.</author><author>Sidow,A.</author></authors></contributors><auth-address>DepartmentofZoology,UniversityofOxford,UK.</auth-address><titles><title>Geneduplicationsandtheoriginsofvertebratedevelopment</title><secondary-title>DevSuppl</secondary-title></titles><pages>125-33</pages><edition>1994/01/01</edition><keywords><keyword>AminoAcidSequence</keyword><keyword>Animals</keyword><keyword>BaseSequence</keyword><keyword>Chordata,Nonvertebrate/genetics</keyword><keyword>*Evolution</keyword><keyword>GeneExpression</keyword><keyword>Genes,Homeobox</keyword><keyword>Genome</keyword><keyword>Insulin/genetics</keyword><keyword>MolecularSequenceData</keyword><keyword>*MultigeneFamily</keyword><keyword>Phylogeny</keyword><keyword>Proto-Oncogenes</keyword><keyword>SequenceHomology</keyword><keyword>Urochordata/genetics</keyword><keyword>Vertebrates/*embryology/genetics</keyword></keywords><dates><year>1994</year></dates><accession-num>7579513</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7579513</url></related-urls></urls><language>eng</language></record></Cite></EndNote>[26]、哺乳类中有4个Hox基因簇。根据以上这些发现，可以对基因倍增时间进行修正，即基因倍增的时间是在头索动物和脊椎动物分化后，在颌口类出现以前。根据从无脊椎动物到无颌类再到哺乳类逐步增加的基因拷贝数，认为发生过两次全基因组倍增事件，在无颌类鱼类出现（500-430Mya）前后各有一次（图1.3），即2R假说ADDINEN.CITE<EndNote><Cite><Author>Holland</Author><Year>1994</Year><RecNum>154</RecNum><record><rec-number>154</rec-number><foreign-keys><keyapp="EN"db-id="xfx2zrs5b2wt5besdpwxts9lv2szdfd5vpsp">154</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Holland,P.W.</author><author>Garcia-Fernandez,J.</author><author>Williams,N.A.</author><author>Sidow,A.</author></authors></contributors><auth-address>DepartmentofZoology,UniversityofOxford,UK.</auth-address><titles><title>Geneduplicationsandtheoriginsofvertebratedevelopment</title><secondary-title>DevSuppl</secondary-title></titles><pages>125-33</pages><edition>1994/01/01</edition><keywords><keyword>AminoAcidSequence</keyword><keyword>Animals</keyword><keyword>BaseSequence</keyword><keyword>Chordata,Nonvertebrate/genetics</keyword><keyword>*Evolution</keyword><keyword>GeneExpression</keyword><keyword>Genes,Homeobox</keyword><keyword>Genome</keyword><keyword>Insulin/genetics</keyword><keyword>MolecularSequenceData</keyword><keyword>*MultigeneFamily</keyword><keyword>Phylogeny</keyword><keyword>Proto-Oncogenes</keyword><keyword>SequenceHomology</keyword><keyword>Urochordata/genetics</keyword><keyword>Vertebrates/*embryology/genetics</keyword></keywords><dates><year>1994</year></dates><accession-num>7579513</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7579513</url></related-urls></urls><language>eng</language></record></Cite></EndNote>[26]。图1.3脊椎动物的全基因组倍增（WGD），基于全基因组分析或Hox基因簇数目，引自ADDINEN.CITE<EndNote><Cite><Author>Panopoulou</Author><Year>2005</Year><RecNum>155</RecNum><record><rec-number>155</rec-number><foreign-keys><keyapp="EN"db-id="zf5r9dwwdfp5p2ef0xk5r2t7dax5tv0e9dsr">155</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Panopoulou,G.</author><author>Poustka,A.J.</author></authors></contributors><auth-address>EvolutionandDevelopmentGroup,DepartmentofVertebrateGenomics,Max-PlanckInstitutfurMolekulareGenetik,Ihnestrasse73,D-14195Berlin,Germany.panopoul@molgen.mpg.de</auth-address><titles><title>Timingandmechanismofancientvertebrategenomeduplications--theadventureofahypothesis</title><secondary-title>TrendsGenet</secondary-title></titles><pages>559-67</pages><volume>21</volume><number>10</number><edition>2005/08/16</edition><keywords><keyword>Animals</keyword><keyword>*Evolution,Molecular</keyword><keyword>Genes,Homeobox/genetics</keyword><keyword>*Genome</keyword><keyword>*Models,Genetic</keyword><keyword>Phylogeny</keyword><keyword>*Polyploidy</keyword><keyword>Vertebrates/*genetics</keyword></keywords><dates><year>2005</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>0168-9525(Print) 0168-9525(Linking)</isbn><accession-num>16099069</accession-num><urls><related-urls><url>/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16099069</url></related-urls></urls><electronic-resource-num>S0168-9525(05)00226-X[pii] 10.1016/j.tig.2005.08.004</electronic-resource-num><language>eng</language></record></Cite></EndNote>[27]。每个不同颜色的圆圈代表一个Hox基因簇。圆圈的排列不代表相应的Hox基因簇在基因组中的排列。箭头指向处表示发生WGD。唯一确切证实的WGD是鱼特有的3RWGD。1R或2RWGD的证据是人类基因组中大量的旁系同源基因和四倍化区域。最近研究无颌类得到的数据表明在其与颌口类分化后有额外的WGD（灰色箭头所示）。对于无颌类是不是单系的现在存在争议(断裂线)ADDINEN.CITE<EndNote><Cite><Author>Takezaki</Author><Year>2003</Year><RecNum>167</RecNum><record><rec-number>167</rec-number><foreign-keys><keyapp="EN"db-id="zf5r9dwwdfp5p2ef0xk5r2t7dax5tv0e9dsr">167</key></foreign-keys><ref-typename="JournalAr