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达氏鲟dmc1和ly75基因的cDNA克隆及在精子生成中的表达分析.docx

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达氏鲟dmc1和ly75基因的cDNA克隆及在精子生成中的表达分析 Title:cDNACloningandExpressionAnalysisofDMC1andLY75GenesintheSpermatogenesisofAcipenserdabryanus Abstract: TheDMC1andLY75genesplayimportantrolesinspermatogenesis,buttheirfunctionsinAcipenserdabryanusremainpoorlyunderstood.Inthisstudy,weclonedthecDNAsequencesofDMC1andLY75genesfromA.dabryanusandanalyzedtheirexpressionpatternsinthetestisduringspermatogenesis. Keywords:Acipenserdabryanus,DMC1,LY75,cDNAcloning,geneexpression,spermatogenesis 1.Introduction Spermatogenesisisacomplexprocessinvolvingthedifferentiationofgermcellsintomaturespermatozoa.Numerousgeneshavebeenfoundtobeessentialfortheproperprogressionofspermatogenesis,includingDMC1andLY75.DMC1isameiosis-specificgeneinvolvedinhomologousrecombination,whileLY75isinvolvedintheprocessesofcelladhesionandmigration.UnderstandingtheexpressionpatternsandfunctionsofthesegenesinA.dabryanuswillprovideinsightsintothemolecularmechanismsunderlyingspermatogenesisinthisspecies. 2.MaterialsandMethods 2.1Samplecollection TestissampleswereobtainedfrommatureA.dabryanusindividualsduringthespawningseason. 2.2cDNAcloning TotalRNAwasextractedfromthetestisusingacommercialRNAextractionkit.ReversetranscriptionwasperformedtoobtaincDNA.PCRprimersdesignedbasedontheconservedregionsofDMC1andLY75geneswereusedtoamplifythetargetcDNAsequences.Theamplifiedfragmentswerepurified,clonedintoasuitablevector,andtransformedintocompetentbacteria.Positivecloneswereselectedandverifiedbysequencing. 2.3Geneexpressionanalysis Quantitativereal-timePCR(qRT-PCR)wasperformedtoanalyzetheexpressionpatternsofDMC1andLY75genesduringdifferentstagesofspermatogenesis.Theexpressionlevelsofthetargetgeneswerenormalizedtoareferencegene,andtherelativegeneexpressionwascalculatedusingthe2^-ΔΔCTmethod.Statisticalanalysiswasconductedtodeterminesignificantdifferencesingeneexpressionlevelsamongdifferentstagesofspermatogenesis. 3.Results 3.1cDNAcloning Thefull-lengthcDNAsequencesofDMC1andLY75genesfromA.dabryanusweresuccessfullyclonedandsequenced.Sequenceal