鳜胃蛋白酶原基因cdna全长的克隆与序列分析

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1、鳜胃蛋白酶原基因cDNA全长的克隆与序列分析吴雪峰，赵金良（上海水产大学农业部水产种质资源与养殖生态重点开放实验室，上海200090）关键词：鳜；胃蛋白酶原；cDNA末端快速扩增；克隆；序列分析中图分类号：S917文献标识码：ACloningandsequencingofthefull-lengthcDNAofpepsinogengenefromtheMandarinfish，SinipercachuatsiWUXue-Feng,ZHAOJin-Liang(KeyLaboratoryofAquaticGeneticResourcesandAquacu

2、lturalEcosystemCertificatedbytheMinistryofAgriculture,ShanghaiFisheriesUniversity,Shanghai200090,China)Abstract:Pepsinogenisonekindofgastricdigestiveproteinasesbelonglingtoafamilyofasparticproteinases,whichissynthesizedinthegastricmucosaofvertebratesandconvertedtopepsinsintheac

3、idicenvironmentofgastricjuice.Themandarinfish(Sinipercachuatsi)isatypicalcarnivorousfishwhichonlypreyonsmalllivefishesafterhatching.Inordertounderstandthemolecularcharactersofpepsinogeninfish,thefull-lengthcDNAofpepsinogengeneofS.chuatsiwasclonedbymeansofRT-PCRandrapidamplifica

4、tionofcDNAends(RACE)andsequenced.Theresultrevealsa1367bpcDNAfull-lengthsequencecontaining43bp5′-untranslatedregion,187bp3′-untranslatedregionand1137bpopenreadingframe(ORF),whichencodes378aminoacidswithcharacteristicsignalpeptidesandactivationpeptides.ThepepsinogenofS.chuatsials

5、ohashighlyconservedaminoacidresiduesessentialforcatalyticactivityandconformationalmaintenance,whicharetwoessentialaspartylresiduesintheactivesiteandsixcysteineresiduesinvolvedinformingthreedisulphidebonds.ThehomologyofaminoacidsequencesbetweenpepsinogenofS.chuatsiandotherverteb

6、ratepepsinogensrangesfrom59.9%to91.2%,whichindicatesthepepsinogengeneishighlyconservedinevolution.TheNJphylogenetictreeofvertebratesfromtheaminoacidssequencesofpepsinogenshowedallfishwereclusteredasagroup,thepepsinogenofS.chuatsiwasthemostclosetopepsinogenAformⅡaofwinterflounde

7、r.ThesuccessfulcloningofpepsinogengenefromS.chuatsinotonlylaysthefoundationforfurtherstudyofitstemporalandspatialexpression,butalsoprovidesnewmaterialforthemolecularcharacteristicandevolutionoffishpepsinogen.Keywords:Sinipercachuatsi;pepsinogen;rapidamplificationofcDNAends;clon

8、ing;sequenceanalysis收稿日期：2007-07-19资助项目：上海市重点学科建设项目(Y1