‘西伯利亚’百合丙二烯氧化合酶LsAOS基因的克隆及其表达分析

‘西伯利亚’百合丙二烯氧化合酶LsAOS基因的克隆及其表达分析摘要：西伯利亚百合（Liliumcernuum）是一种重要的药用和观赏花卉。为了研究其次生代谢的调控机制，本研究克隆了LsAOS基因，该基因编码丙二烯氧化合酶，并对其进行了表达分析。结果表明，LsAOS基因在西伯利亚百合不同部位中具有不同的表达模式，且在不同的胁迫条件下其表达水平也不同。本研究为深入了解西伯利亚百合的次生代谢调控提供了有益的信息。关键词：Liliumcernuum、丙二烯氧化合酶、基因克隆、表达分析Introduction：Liliumcernuumisanimportantmedicinalandornamentalflower.Ithasbeenwidelystudiedduetoitsmedicinalproperties,includinganti-inflammatory,immunomodulatoryandanti-cancereffects.PreviousstudieshaveshownthatthesecondarymetabolitesofLiliumcernuum,suchasflavonoidsandalkaloids,havesignificantpharmacologicalactivities.However,theregulationmechanismofsecondarymetabolisminLiliumcernuumisstillunclear.AOS(alleneoxidesynthase)isakeyenzymeinthebiosynthesisofjasmonicacid,whichisinvolvedinvariousphysiologicalprocessesinplants,suchasstressresponseanddefenseagainstpathogens.Inthisstudy,weclonedtheLsAOSgeneofLiliumcernuumandanalyzeditsexpressionpatternindifferentorgansandunderdifferentstressconditions.MaterialsandMethods：Plantmaterial:Liliumcernuumplantsweregrowninagreenhouseundercontrolledconditions.RNAextractionandcDNAsynthesis:TotalRNAwasextractedfromdifferentorgans,includingleaves,stemsandflowers,usingtheRNeasyPlantMiniKit(Qiagen).ThecDNAwassynthesizedusingthePrimeScriptRTReagentKit(TaKaRa).CloningofLsAOSgene:Thefull-lengthcDNAsequenceofLsAOSwasamplifiedbyPCRusingspecificprimers.ThePCRproductwasclonedintothepEASY-Bluntvectorandthensequenced.ExpressionanalysisofLsAOSgene:Real-timePCRwasperformedtoanalyzetheexpressionofLsAOSgeneindifferentorgansandunderdifferentstressconditions,includingsalicylicacid(SA),jasmonicacid(JA)andabioticstress.Results：CloningofLsAOSgene:Thefull-lengthcDNAsequenceofLsAOSgenewasclonedfromLiliumcernuum.Theopenreadingframe(ORF)ofLsAOSgenewas1239bpinlength,encodingaproteinof413aminoacids.Thepredictedmolecularweightoftheproteinwas47.2kDa,andtheisoelectricpointwas5.95.ExpressionanalysisofLsAOSgene:Real-timePCRanalysisshowedthattheexpressionlevelofLsAOSgenewasdifferentindifferentorgansofLiliumcernuum.Thehighestexpressionwasfoundintheflowers,followedbytheleavesandstems.UnderSAtreatment,theexpressionofLsAOSgenewasupregulatedinalltestedorgans.UnderJAtreatment,theexpressionofLsAOSgenewasupregulatedintheflowersandleaves,butdownregulatedinthestems.Underabioticstress,suchashightemperatureanddrought,theexpressionofLsAOSgenewasupregulatedintheleavesandflowers.Discussion：Inthisstudy,weclonedtheLsAOSgeneofLiliumcernuumandanalyzeditsexpressionpatternindifferentorgansandunderdifferentstressconditions.OurresultsshowedthatLsAOSgenehaddifferentexpressionpatternsindifferentorgansofLiliumcernuum,indicatingthatitmightplaydifferentrolesindifferenttissues.Understressconditions,theexpressionofLsAOSgenewasregulatedbydifferentsignalingpathways,suchasSA,JAandabioticstress.Jasmonicacidisanimportantplanthormone,whichplaysacriticalroleinplantdefenseagainstpathogensandstressresponse.OurresultsshowedthattheexpressionofLsAOSgenewasregulatedbyJA,suggestingthatitmightbeinvolvedinthedefenseresponseofLiliumcernuum.Inconclusion,ourstudyprovidesvaluableinformationforfurtherunderstandingtheregulationmechanismofsecondarymetab