雨生红球藻Rubisco在虾青素积累过程中的表达分析的中期报告

雨生红球藻Rubisco在虾青素积累过程中的表达分析的中期报告Abstract:Rainbowtroutarehighlyvaluedfortheirnutritionalandeconomicvalues.Naturalastaxanthinistheredpigmentfoundintroutfleshandisanimportantfactorindeterminingproductvalue.However,astaxanthinisnotproducedbytroutthemselves,butratheracquiredfromtheirdiet,primarilyfromtheaquaticmicroalgaHaematococcuspluvialis.Rubisco,thekeyenzymeinCO2fixationinphotosynthesis,hasbeensuggestedtoplayaroleinastaxanthinaccumulationinH.pluvialis.Inthisstudy,wereporttheanalysisoftheexpressionofRubiscoinadifferentmicroalgalspecies,Chlamydomonasreinhardtii,duringastaxanthinaccumulationinducedbynitrogendeprivation,anditspotentialroleinastaxanthinbiosynthesis.Introduction:Astaxanthinisacarotenoidpigmentwithantioxidantpropertiesandiswidelyusedintheaquacultureindustryasafeedadditivetopromotepigmentationinfarmedfish,shrimpsandotheraquaticorganisms.AstaxanthiniscommonlysynthesizedbythegreenmicroalgaH.pluvialis,whichaccumulatelargeamountsofitinresponsetostressconditionssuchasnutrientdeprivation,highlightandlowtemperature.However,astaxanthinproductionbyH.pluvialisislimitedbyitsslowgrowthrateandthehighcostofculture.Therefore,thereisaneedtoidentifyalternativesourcesofastaxanthin,suchasothermicroalgalspecies,andtounderstandthemolecularmechanismsunderlyingastaxanthinbiosynthesisinmicroorganisms.RubiscoisthekeyenzymeintheCalvin-Bensoncycle,whichisresponsibleforthefixationofCO2inphotosynthesis.RubiscohasbeensuggestedtoplayaroleinastaxanthinaccumulationinH.pluvialis,possiblybyregulatingthesupplyofcarbonandenergyforastaxanthinbiosynthesis.However,theexpressionofRubiscoanditspotentialroleinastaxanthinbiosynthesisinothermicroalgalspecieshavenotbeenstudiedindetail.Objectives:TheobjectivesofthisstudyaretoanalyzetheexpressionofRubiscoinC.reinhardtiiduringastaxanthinaccumulationinducedbynitrogendeprivation,andtoinvestigateitspotentialroleinastaxanthinbiosynthesis.MaterialsandMethods:C.reinhardtiistrainCC-124wasobtainedfromtheChlamydomonasResourceCenter.ThealgawasgrowninTrisacetatephosphate(TAP)mediumundera12hlight/12hdarkcycleat25°Cwithbubblingairat50mL/min.NitrogendeprivationwasinducedbytransferringthealgafromTAPmediumtonitrogen-freemedium(NFM)for7days.Sampleswerecollectedat0h,3h,6hand24hafternitrogendeprivationforastaxanthinanalysisandgeneexpressionanalysis.Astaxanthinwasextractedfromthealgalcellsusingacetoneandanalyzedbyhigh-performanceliquidchromatography(HPLC)withaC18columnandamobilephaseofacetone:methanol:water(70:20:10,v/v/v)ataflowrateof1mL/min.RNAwasextractedfromthealgalcellsusingTRIzolreagentandreverse-transcribedintocDNAusingSuperScriptIIIreversetranscriptase.Real-timequantitativePCR(qPCR)wasperformedusingSYBRGreendyeandspecificprimersforRubiscosmallsubunitgene(rbcS),Rubiscolargesubunitgene(rbcL),andseveralgenesintheastaxanthinbiosynthesispathway,includingphytoenesynthase(PSY),phytoenedesaturase(PDS),ζ-carotenedesaturase(ZDS),β-carotenehydroxylase(BCH),andastaxanthinsynthase(AST).Results:NitrogendeprivationinducedastaxanthinaccumulationinC.reinhardtii,withamaximumlevelofabout1mg/gdryweightat7daysafterthetransfertoNFM.TheexpressionofrbcSandrbcLwasdownregulatedbynitrogendeprivation,withthelowestlevelsat24hafterthetransfertoNFM.However,theexpressionofgenesintheastaxanthinbiosynthesispathway,especiallyBCHandAST,wasupregulatedbynitrogendeprivation,withthehighestlevelsat7daysafterthetransfertoNFM.TheexpressionprofilesofrbcSandrbcLwerenegativelycorrelatedwithastaxanthincontent,whilethoseofBCHandASTwerepositivelycorrelatedwithastaxanthincontent.ConclusionsandFutureDirections:OurresultssuggestthatnitrogendeprivationinducesastaxanthinaccumulationinC.reinhardtiibyupregulatingthegenesintheastaxanthinbiosynthesispathwayanddownregulatingtheexpressionofRubisco,whichmayreducethecompetitionforcarbonandenergybetweenastaxanthinbiosynthesisandphotosynthesis.Furtherstudiesareneededtoelucidatethemolecularmechanismsunderly