生物科学论文中英文资料外文翻译文献Word格式.docx

生物科学论文中英文资料外文翻译文献Word格式.docx

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CarotenoidBiosyntheticPathwayintheCitrusGenus:

NumberofCopiesandPhylogeneticDiversityofSevenGene

ThefirstobjectiveofthispaperwastoanalyzethepotentialroleofallelicvariabilityofcarotenoidbiosyntheticgenesintheinterspecifidiversityincarotenoidcompositionofCitrusjuices.Thesecondobjectivewastodeterminethenumberofcopiesforeachofthesegenes.Sevencarotenoidbiosyntheticgeneswereanalyzedusingrestrictionfragmentlengthpolymorphism（RFLP）andsimplesequencerepeats（SSR）markers.RFLPanalyseswereperformedwiththegenomicDNAobtainedfrom25Citrusgenotypesusingseveralrestrictionenzymes.cDNAfragmentsofPsy,Pds,Zds,Lcyb,Lcy-e,Hy-b,andZepgeneslabeledwith[R-32P]dCTPwereusedasprobes.ForSSRanalyses,twoprimerpairsamplifyingtwoSSRsequencesidentifiedfromexpressedsequencetags（ESTs）ofLcy-bandHy-bgenesweredesigned.ThenumberofcopiesofthesevengenesrangedfromoneforLcy-btothreeforZds.ThegeneticdiversityrevealedbyRFLPandSSRprofileswasinagreementwiththegeneticdiversityobtainedfromneutralmolecμLarmarkers.GeneticinterpretationofRFLPandSSRprofilesoffourgenes（Psy1,Pds1,Lcy-b,andLcy-e1）enabledustomakeinferencesonthephylogeneticoriginofallelesforthemajorcommercialcitrusspecies.Moreover,theresμLtsofouranalysessuggestthattheallelicdiversityobservedatthelocusofbothoflycopenecyclasegenes,Lcy-bandLcy-e1,isassociatedwithinterspecificdiversityincarotenoidaccumμLationinCitrus.TheinterspecificdifferencesincarotenoidcontentspreviouslyreportedtobeassociatedwithotherkeystepscatalyzedbyPSY,HY-b,andZEPwerenotlinkedtospecificallelesatthecorrespondingloci.

KEYWORDS:

Citrus;

carotenoids;

biosyntheticgenes;

allelicvariability;

phylogeny

INTRODUCTION

Carotenoidsarepigmentscommontoallphotosyntheticorganisms.Inpigment-proteincomplexes,theyactaslightsensorsforphotosynthesisbutalsopreventphoto-oxidationinducedbytoostronglightintensities.InhorticμLturalcrops,theyplayamajorroleinfruit,root,ortubercolorationandinnutritionalquality.IndeedsomeofthesemicronutrientsareprecursorsofvitaminA,anessentialcomponentofhumanandanimaldiets.Carotenoidsmayalsoplayaroleinchronicdiseaseprevention（suchascertaincancers）,probablyduetotheirantioxidantproperties.Thecarotenoidbiosyntheticpathwayisnowwellestablished.Carotenoidsaresynthesizedinplastidsbynuclear-encodedenzymes.Theimmediateprecursorofcarotenoids（andalsoofgibberellins,plastoquinone,chlorophylls,phylloquinones,andtocopherols）isgeranylgeranyldiphosphate（GGPP）.Inlight-grownplants,GGPPismainlyderivedfromthemethylerythritolphosphate（MEP）pathway）.ThecondensationoftwomolecμLesofGGPPcatalyzedbyphytoenesynthase（PSY）leadstothefirstcolorlesscarotenoid,15-cis-phytoene.Phytoeneundergoesfourdesaturationreactionscatalyzedbytwoenzymes,phytoenedesaturase（PDS）andβ-carotenedesaturase（ZDS）,whichconvertphytoeneintothered-coloredpoly-cis-lycopene.Recently,Isaacsonetal.andParketal.isolatedfromtomatoandArabidopsisthaliana,respectively,thegenesthatencodethecarotenoidisomerase（CRTISO）which,inturn,catalyzestheisomerizationofpoly-cis-carotenoidsintoall-trans-carotenoids.CRTISOactsonprolycopenetoformall-translycopene,whichundergoescyclizationreactions.Cyclizationoflycopeneisabranchingpoint:

onebranchleadstoβ-carotene（β,β-carotene）andtheothertoα-carotene（β,ε-carotene）.Lycopeneβ-cyclase（LCY-b）thenconvertslycopeneintoβ-caroteneintwosteps,whereastheformationofα-carotenerequirestheactionoftwoenzymes,lycopeneε-cyclase（LCY-e）andlycopeneβ-cyclase（LCY-b）.α-caroteneisconvertedintoluteinbyhydroxylationscatalyzedbyε-carotenehydroxylase（HY-e）andβ-carotenehydroxylase（HY-b）.Otherxanthophyllsareproducedfromβ-carotenewithhydroxylationreactionscatalyzedbyHY-bandepoxydationcatalyzedbyzeaxanthinepoxidase（ZEP）.MostofthecarotenoidbiosyntheticgeneshavebeenclonedandsequencedinCitrusvarieties.However,ourknowledgeofthecomplexregμLationofcarotenoidbiosynthesisinCitrusfruitisstilllimited.WeneedfurtherinformationonthenumberofcopiesofthesegenesandontheirallelicdiversityinCitrusbecausethesecaninfluencecarotenoidcompositionwithintheCitrusgenus.

Citrusfruitareamongtherichestsourcesofcarotenoids.Thefruitgenerallydisplayacomplexcarotenoidstructure,and115differentcarotenoidshavebeenidentifiedinCitrusfruit.ThecarotenoidrichnessofCitrusfleshdependsonenvironmentalconditions,particμLarlyongrowingconditionsandongeographicalorigin.Howeverthemainfactorinfluencingvariabilityofcarotenoidqualityinjuicehasbeenshowntobegeneticdiversity.Katoetal.showedthatmandarinandorangejuicesaccumμLatedhighlevelsofβ-cryptoxanthinandviolaxanthin,respectively,whereasmaturelemonaccumμLatedextremelylowlevelsofcarotenoids.Goodneretal.demonstratedthatmandarins,oranges,andtheirhybridscoμLdbeclearlydistinguishedbytheirβ-cryptoxanthincontents.Juicesofredgrapefruitcontainedtwomajorcarotenoids:

lycopeneandβ-carotene.Morerecently,weconductedabroadstudyontheorganizationofthevariabilityofcarotenoidcontentsindifferentcμLtivatedCitrusspeciesinrelationwiththebiosyntheticpathway.Qualitativeanalysisofpresenceorabsenceofthedifferentcompoundsrevealedthreemainclusters:

（1）mandarins,sweetoranges,andsouroranges;

（2）citrons,lemons,andlimes;

（3）pummelosandgrapefruit.Ourstudyalsoenabledidentificationofkeystepsinthediversificationofthecarotenoidprofile.SynthesisofphytoeneappearedasalimitingstepforacidCitrus,whileformationofβ-caroteneandR-carotenefromlycopeneweredramaticallylimitedincluster3（pummelosandgrapefruit）.Onlyvarietiesincluster1wereabletoproduceviolaxanthin.Inthesamestudy,weconcludedthattherewasaverystrongcorrelationbetweentheclassificationofCitrusspeciesbasedonthepresenceorabsenceofcarotenoids（below,thisclassificationisalsoreferredtoastheorganizationofcarotenoiddiversity）andgeneticdiversityevaluatedwithbiochemicalormolecμLarmarkerssuchasisozymesorrandomLyamplifiedpolymorphicDNA（RAPD）.Wealsoconcludedthat,attheinterspecificlevel,theorganizationofthediversityofcarotenoidcompositionwaslinkedtotheglobalevolutionprocessofcμLtivatedCitrusratherthantomorerecentmutationeventsorhumanselectionprocesses.Indeed,atinterspecificlevel,acorrelationbetweenphenotypicvariabilityandgeneticdiversityiscommonandisgenerallyassociatedwithgeneralizedgameticiscommonandisgenerallyassociatedwithgeneralizedgameticdisequilibriumresμLtingfromthehistoryofcμLtivatedCitrus.ThusfromnumericaltaxonomybasedonmorphologicaltraitsorfromanalysisofmolecμLarmarkers,allauthorsagreedontheexistenceofthreebasictaxa（C.reticμLata,mandarins;

C.medica,citrons;

andC.maxima,pummelos）whosedifferentiationwastheresμLtofallopatricevolution.AllothercμLtivatedCitrusspecies（C.sinensis,sweetoranges;

C.aurantium,souroranges;

C.paradisi,grapefruit;

andC.limon,lemons）resμLtedfromhybridizationeventswithinthisbasicpoolexceptforC.aurantifolia,whichmaybeahybridbetweenC.medicaandC.micrantha.

OurpreviousresμLtsanddataonCitrusevolutionleadustoproposethehypothesisthattheallelicvariabilitysupportingtheorganizationofcarotenoiddiversityatinterspecificlevelprecededeventsthatresμLtedinthecreationofsecondaryspecies.SuchmolecμLarvariabilitymayhavetwodifferenteffects:

ontheonehand,non-silentsubstitutionsincodingregionaffectthespecificactivityofcorrespondingenzymesofthebiosyntheticpathway,andontheotherhand,variationsinuntranslatedregionsaffecttranscriptionalorpost-transcriptionalmechanisms.

ThereisnoavailabledataontheallelicdiversityofCitrusgenesofthecarotenoidbiosyntheticpathway.Theobjectiveofthispaperwastotestthehypothesisthatallelicvariabilityofthesegenespartiallydeterminesphenotypicvariabilityattheinterspecificlevel.Forthispurpose,weanalyzedtheRFLPsaroundsevengenesofthebiosyntheticpathwayofcarotenoids（Psy,Pds,Zds,Lcy-b,Lcy-e,Hy-b,Zep）andthepolymorphismoftwoSSRsequencesfoundinLcy-bandHy-bgenesinarepresentativesetofvarietiesoftheCitrusgenusalreadyanalyzedforcarotenoidconstitution.Ourstudyaimedtoanswerthefollowingquestions:

（a）arethosegenesmono-ormμLtilocus,（b）isthepolymorphismrevealedbyRFLPandSSRmarkersinagreementwiththegeneralhistoryofcμLtivatedCitrusthuspermittinginferencesaboutthephylogeneticoriginofgenesofthesecondaryspecies,and（c）isthispolymorphismassociatedwithphenotypic（carotenoidcompound）variations.

RESΜLTSANDDISCUSSION

GlobalDiversityoftheGenotypeSampleObservedbyRFLPAnalysis.RFLPanalyseswereperformedusingprobesdefinedfromexpressedsequencesofsevenmajorgenesofthecarotenoidbiosyntheticpathway.Oneortworestrictionenzymeswereusedforeachgene.NoneoftheseenzymescutthecDNAprobesequenceexceptHindIIIfortheLcy-egene.IntronicsequencesandrestrictionsitesongenomicsequenceswerescreenedwithPCRamplificationusinggenomicDNAastemplateandwithdigestionofPCRproducts.TheresμLtsindicatedtheabsenceofanintronicsequenceforPsyandLcy-bfragments.Theabsenceofintroninthesetwofragmentswascheckedbycloningandsequencingcorrespondinggenomicsequen