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家蚕核型多角体病毒编码circRNAcirc_10功能研究.docx

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家蚕核型多角体病毒编码circRNAcirc_10功能研究 Title:FunctionalInvestigationofcircRNAcirc_10EncodedbytheBombyxmoriNuclearPolyhedrosisVirusinSilkworms Abstract: CircularRNAs(circRNAs)haveemergedasanovelclassofnon-codingRNAswithdiversebiologicalfunctions.Inthisstudy,wefocusoncirc_10,acircRNAencodedbytheBombyxmoriNuclearPolyhedrosisVirus(BmNPV),andinvestigateitspotentialroleinthesilkwormhost.Utilizingbothmolecularandfunctionalapproaches,weprovideinsightsintothemechanismbywhichcirc_10modulateshostgeneexpressionandcontributestothepathogenesisofBmNPV. 1.Introduction: Silkworms(Bombyxmori)areeconomicallyimportantinsectsthatprovidevaluablesilkproductionandserveasmodelsforstudyingviralinfections.TheBmNPVisacommonviralpathogenofsilkworms,causingsignificanteconomiclossesinsericulture.RecentstudieshavehighlightedtheinvolvementofcircRNAsinregulatingvariousbiologicalprocesses,includingviralpathogenesis.However,thefunctionalcharacterizationofcircRNAsencodedbyviralgenomes,suchascirc_10inBmNPV,remainslargelyunexplored. 2.Methods: 2.1Identificationandcharacterizationofcirc_10:Weemployedbioinformaticstoolstoidentifyandannotatecirc_10,followedbyexperimentalvalidationusingRT-PCRandSangersequencing.Thecircularnatureofcirc_10wasconfirmedbyresistancetoRNaseRdigestionandverificationoftheback-splicingjunction. 2.2Expressionanalysisofcirc_10:Quantitativereal-timePCRwasperformedtoexaminetheexpressionpatternofcirc_10duringdifferentstagesofBmNPVinfection.Fluorescenceinsituhybridization(FISH)wasusedtodeterminethesubcellularlocalizationofcirc_10. 2.3Assessmentofcirc_10interactionwithhostfactors:RNAimmunoprecipitation(RIP)assayswerecarriedouttoidentifypotentialbindingpartnersofcirc_10.RNApulldownandmassspectrometryanalysiswereperformedtoinvestigatethefunctionalrelevanceofcirc_10-hostproteininteractions. 3.Results: 3.1Characterizationofcirc_10:Weidentifiedcirc_10asahighlystableandabundantcircRNAgeneratedfromBmNPVgenome.Sangersequencingconfirmedtheback-splicingjunctionandcircularstructureofcirc_10. 3.2Expressionanaly