MolecularcloningandexpressionanalysisofglutathioneperoxidaseandglutathionereductasefromGracilarialemaneiformisunderheatstress
NingLu&YanDing&Xiao-NanZang&Xue-ChengZhang&HaoChen&Xiao-ShengMu
Received:1November2012/Revisedandaccepted:4March2013/Publishedonline:23March2013#SpringerScience+BusinessMediaDordrecht2013
AbstractGlutathioneperoxidase(GPx)andglutathionereductase(GR)arekeyenzymesintheantioxidantdefensesystemsoflivingorganismsandprotectorgan-ismsfromenvironmentalstresses.ToinvestigatethemechanismofresistancetoheatstressintheredalgaGracilarialemaneiformis,thecDNAsequencesencodingglutathioneperoxidaseandglutathionereductaseoftwostrains(wildtypeandheat-tolerantcultivar981)ofG.lemaneiformisweresuccessfullyclonedusingreversetranscriptionPCRandrapidamplificationcDNAendstechniques.cDNAencodingGPxhas930nucleotideswithanopenreadingframe(ORF)of750nucleotides,encoding249aminoacidresidues.cDNAencodingGRwas1,572nucleotideswitha1,416-nucleotideORFencoding471aminoacids.Nointronsexistedingeno-micDNAofGPxandGRofG.lemaneiformis.South-ernblottingindicatedthattherewasonlyonecopyofGPxandGRinbothstrains.Underheatstress,bioac-tivityandmRNAtranscriptionlevelsofbothGPxandGRwereupregulatedincultivar981comparedtothoseinthewildtype.TheseresultsindicatethatbothGPxandGRmayplayimportantrolesintheabilityofG.lemaneiformistoresisthightemperatures.
KeywordsExpressionanalysis.Genecloning.Glutathioneperoxidase.Glutathionereductase.Gracilarialemaneiformis.Rhodophyta.Heatstress
N.Lu:Y.Ding:X.-N.Zang(*):X.-C.Zhang:H.Chen:X.-S.Mu
KeyLaboratoryofMarineGeneticsandBreeding,MinistryofEducation,OceanUniversityofChina,Qingdao266003Shandong,China
e-mail:xnzang@ouc.edu.cn
Introduction
TheredalgaGracilarialemaneiformis(BorydeSaint-Vincent)Grevilleiseconomicallyimportantasanagarophyteandasafeedstuffforabaloneaquaculture,aswellasbeinganidealmaterialforgeneticstudies(Chenetal.2009).Inrecentyears,itscultivationhasdevelopedextensively,andithasbecomethethirdmostcultivatedalgafollowingLaminariaandPorphyrainChina.Whilethesuitablegrowthtempera-tureofthewildpopulationisbetween11and23°C,itcannotendurehighsummertemperaturesorlowwintertemperatures,andthus,itsgrowthseasonisrelativelyshort.Inordertosolvethisproblem,researchershavebredanewstrainofG.lemaneiformisfromthewildpopulation,“cultivar981,”whichcanenduretemperaturesupto26°Candhashighproductivitywithahighconcentrationofagar(Guetal.2012;Luetal.2012).Despitethissuperiorquality,cultivar981cannotsurvivethroughthesummeralongthesouthcoastsofChina,andso,itisnecessarytobreednewstrainsthatcanendureevenhighertemperatures.Therefore,inordertobreedastrainresistanttodamagebyhighertemperatures,itisnecessarytostudythemechanismsofresponsetoheatstressofG.lemaneiformis.
Theantioxidantenzymaticsystemplaysanimportantroleinresistingheatstress(Zhangetal.2010).Glutathioneperoxidase(GPx)andglutathionereductase(GR)arekeyenzymesintheantioxidantdefensesystemsoflivingorgan-ismsandarethoughttoprotectvariousorganismsfromoxidativestresses(Allaetal.2007).GPxcatalyzesthereductionofH2O2,organichydroperoxides,andlipidhy-droperoxidesusingGSHasareducingagent.Thishelpstoprotectthecellsagainstoxidativedamage.Simultaneously,thereducedformofGSHisoxidatedtoglutathionedisulfide(GSSG)(Giblin2000).GRinconjunctionwithNADPH
1926catalyzesthereductionofGSSGtoGSHandparticipatesintheremovalofH2O2viatherecyclingoftheGSHpoolwhichiscentraltocounteractingoxidativestress(MullineauxandCreissen1997).Todate,theDNAandcDNAencodingGPxandGRhavebeendeterminedinmanyorganisms,includingNicotianatabacum(Criquietal.1992;CreissenandMullineaux1995),Arabidopsisthaliana(SugimotoandSakamoto1997),Oryzasativa(Lietal.2000;Kaminakaetal.1998),andsomealgae,suchasChlamydomonassp.ICE-L(Dingetal.2012),Scenedesmusquadricauda(Vítováetal.2011),andChlamydomonasreinhardtii(Leisingeretal.1999).However,theDNA/cDNAencodingforG.lemaneiformishasyettobediscovered.
Inourpreviouswork,thetranscriptsinG.lemaneiformisthatlivedundernormalconditionsandunderheatstresswerecomparedusingsuppressionsubtractivehybridization(SSH)fortheidentificationdifferentialexpressedgenes.GPxandGRwereidentifiedamongthesedifferentiallyexpressedgenes.InordertounderstandtherelationshipsofGPxandGRactivitiesandtheirgeneexpressionsinresponsetoheatstressinG.lemaneiformis,weclonedthefullcDNAandDNAsequencesencodingGPxandGRandthenanalyzedthemRNAtranscriptionandactivitiesofGPxandGRintwostrainsofG.lemaneiformis—thewildtypeandthecultivar981underheatstress.
Materialsandmethods
Thecultivar981ofG.lemaneiformiswascollectedfromacultivationfieldatNanaoIslandinShantou,China(23.4°N,117.0°E).ThewildtypeofG.lemaneiformiswasobtainedfromZhanshanBayinQingdao,China(36.0°N,120.2°E).Thealgaewerethoroughlyrinsedwithsterilizedseawaterandculturedinsterilizednaturalseawatersupplementedwith100μMNaNO3and10μMNaH2PO4H2O(finalconcentration)at20±1°C.Intensityofilluminationwas50–60μmolphotonsm−2s−1foralight/darkperiodof12:12.Afteramonth,alltheseaweedsgrewwell,andtheywereusedfortheexperiments.
TotalRNAisolation,cDNAsynthesis,andcloningofGPxandGRgenes
TotalRNAwasisolatedfrom200mgfreshalgaeofcultivar981andwildtypeofG.lemaneiformis,respectively,basedontheintroductionofRNApreppureplantkit(TIANGEN,China).TheRNAswerequantifiedbyspectrophotometerandverifiedbyrunningsampleson1.0%agarosegels;1μgtotalRNAwassubjectedtocDNAsynthesisbySuperScript™IIreversetranscription(RT)-PCRkit(TaKaRa,China)in25μLvolumeaccordingtoinstructionmanual,andanoligo(dT)adaptor
JApplPhycol(2013)25:1925–1931
primer(5′-AAGCAGTGGTATCAACGCAGAGTACT-3′)wasusedastheRTprimertointroduceanadaptor.ThecDNAobtainedwasusedforgenecloningandreal-timePCR.
WeinitiallyobtainedpartialcDNAsequencesofGPxandGRfromtheSSHlibrariesofcultivar981andthewildtypeunderheatstress.AccordingtotheknowncDNAsequenceofGPx,primersGPx-1(5′-AGAAGCAGTTTGGTGCGAAGTT-3′)andGPx-2(5′-TGCAAAAGCACAGATCTCGTCGTTC-3′)weredesignedfor5′and3′rapidamplificationofcDNAends(RACE)ofGPx,respectively.AccordingtotheknowncDNAsequenceofGR,primersGR-1(5′-GTGATCGGTGCAGGATCTGGAGGTGT-3′)andGR-2(5′-CATCCGACATTGACACAAGTACCACCCA-3′)weredesignedfor5′and3′RACEofGR,respectively.Thefull-lengthcDNAsequenceswereobtainedbyoverlappingthreecDNAfragments:the5′end,3′end,andcoreregionofthecDNA.Homologyanalysisofthesequencewasperformedusingthebasiclocalalignmentsearchtool(BLAST)programfromtheNCBIdatabases(Altschuletal.1990).TheoreticalisoelectricpointandmolecularweightwerepredictedusingExPASyProtParamtools(http://us.expasy.org/tools/protparam.html).MultiplesequenceswerealignedusingtheClustalX1.81program(Thompsonetal.1997).Thefinalphylogenetictreeswereconstructedbyneighbor-joiningalgorithmsofMEGA3.1(Kumaretal.2004).Bootstrappingwasperformed1,000timestoobtainsupportvaluesforeachbranch.
CloningofgenomicDNAofGPxandGRandgenomicSouthernblotting
GenomicDNAofbothofcultivar981andwildtypeofG.lemaneiformiswereextractedusingtheUniversalGenomicDNAExtractionkit(TaKaRa,China).BasedonthecDNAsequencesofGPxandGR,wedesignedspecificprimers—GPx-3(5′-ATGAGTTGCTCGTTCCTTGGTCTT-3′),GPx-4(5′-TTATGAGACACCGAGTGACGGCTTT-3′),GR-3(5′-ATGGCTACAACGACCGACCAAT-3′),andGR-4(5′-TTACTGGGGGTTTGGTTCTTTGGT-3′)—toclonetheDNAsequencesofGPxandGR,respectively,incultivar981andwildtype.
DNAfromthewildtypeandcultivar981(2.5μg)wasdigestedwithtwoendonucleasemixtures(each0.5U)for45min,andthen,thefragmentswereseparatedina0.8%agarosegelandtransferredontoanylonmembrane.ForGPx,bothofthewildtypeandcultivar981weredigestedwithrestrictionendonucleasemixture1ofKpnIandEcoRIandrestrictionendonucleasemixture2ofSacIandBamHI.ForGR,bothofthewildtypeandcultivar981weredigestedwithrestrictionendonucleasemixture1ofNotIandBamHIandrestrictionendonucleasemixture2ofNcoIandXhoI.Alltheendonucleaseswerechosenas“non-cutenzymes”forGPxorGRbyDNAMAN.Thefull-length
JApplPhycol(2013)25:1925–1931DNAofGPxorGRwasthenlabeledbyDIGtobeusedastheprobe.TheprobeandthegenomicDNAfragmentsontheblotwerecultivatedat42°Cforhybridization.Afterwashingthemembranetwice(2×15min)withwashingbuffer,thehybridizedfragmentscanbevisual-izedwiththecolorimetricsubstratesNBT/BCIP.TheproceduresforhybridizationandexposurewereaccordingtotheDIG-HighPrimeDNALabelingandDetectionStarterkitI(Roche,Germany)forSouthernblotanalysis.Throughthebandsnumberonthemem-brane,thecopynumberofthetargetgeneingenomicDNAcouldbespeculated.
ActivityassaysandrelativetranscriptiondetectionsofGPxandGRunderheatstress
GPxandGRactivitieswereassayedwhenG.lemaneiformishadbeensubjectedtoaheatstressof32°Cfor0,24,48,and72h,respectively.Thecrudeenzymeextractswerepreparedusing0.5gsamplesin5mLextractionbuffer(0.1molL−1phosphatebuffer,pH7.5).Thehomogenatewascentrifugedat1,000×gfor10minat4°C,andthe
Fig.1cDNAandaminoacidssequencesofGPxgeneofG.lemaneiformis.UnderlineindicatesthestartcodonofATGandthestopcodonofTAA;boxindicatesconservedactiveresidues;doubleunderlineindicatesGPxfamilysignaturemotif
1927
supernatantswerethenusedfortheenzymeassays.TheactivitieswereestimatedaccordingtotheinstructionmanualoftheGPxandGRdetectionkits(Jiancheng,China).
Real-timePCRanalysisoftheGPxandGRmRNArelativetranscriptionlevelsathightemperatures(32°C)wereperformedusingRealMasterMix(SYBRGreen)
Fig.2cDNAandaminoacidssequencesofGRgeneofG.lemaneiformis.UnderlineindicatesthestartcodonofATGandthestopcodonofTAA;doubleunderlineindicatestheredox-activedisul-fidebridge;dottedunderlineindicatestheglutathione-bindingresi-dues,andboxindicatestheconservedresiduesforGSSGbinding
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(TIANGEN,China)andanABI7500Fastreal-timePCRplatform.Bothstrainsofwildtypeandcultivar981ofG.lemaneiformishadbeensubjectedtoaheatstressof32°Cfor0,6,12,24,48,and72h.ToremovetheremaininggenomicDNA,RNAwastreatedwithDNaseI(30U)for15min.TheRNAextractionforallsampleswasfixedatthesametime(2pm)toavoiddifferencesintranscriptionlevelsofGPxandGRcausedbythelightcycle.The18SrRNAwasusedasaninternalcontrolandtheprimers18sF(5′-TGGTGGAGTGATCTGTCTGGTT-3′)and18sR(5′-TTGGCCCGTTCAGTGTAGC-3′)weredesignedaccordingtothe18SrRNAgene(EU561239.1).Thespe-cificprimers—GPx-5(5′-CGCTTCACGACGAATATCA-3′)/GPx-6(5′-TAAAGGGGAGCAACACCAC-3′)andGR-5(5′-GCTGCTGGTGAGGTTATT-3′)/GR-6(5′-GGTTTGGTTCTTTGGTCC-3′)—weredesignedforRT-PCRaccordingtothefull-lengthcDNAsequencesofGPxandGR.TheRT-PCRprocessconsistedofonecycleof2minat95°Candthen40cyclesof15sat95°Cand45sat60°C,followedby30sat68°C.Thedatawerecollectedattheendofeachextensionstep.Therelativetranscriptionsofgenesforeachtreatmentgroupwereanalyzedbythe2−ΔΔCtmethod(LivakandSchmittgen2001).
AllthedatawereanalyzedusingStudent'sttestfortwo-tailedarraysusingMicrosoftExcel,version7.0.
ofGPxis930nucleotides,whichcontaineda5′untranslatedregion(UTR)of109nucleotides,a750-nucleotideopenreadingframe(ORF),and71nucleotidesof3′UTRcontainingapoly(A)tail.TheGCcontentofthefullcDNAofGPxis49.4%.TheORF,startingwithanATGatposition110andendingwithaTAAatposition859,en-codesaproteinof249aminoacidswiththetheoreticalisoelectricpointof8.22andapredictedmolecularweightof27.51kDa.
ThenucleotideanddeducedaminoacidsequencesofGRareshowninFig.2.ThecompletesequenceofGRcDNAis1,572nucleotideslongandcontainedanORFof1,416nucleotides,120nucleotidesof5′UTRupstreamofthestartcodon(ATG),and36nucleotidesof3′UTRwithapoly(A)tailfollowingthestopcodon(TAA).TheORFisfrom121thto1,536thbasepair,whichencodesapolypeptideof471aminoacids,withacalculatedmolecularmassof51.86kDaandatheoreticalisoelectricpointof7.42.TheGCcontentofthecompletecDNAofGRis46.6%.
HomologyanalysisandphylogeneticanalysisofGPxandGR
BLASTandphylogeneticanalysisyieldedthatGPxofG.lemaneiformishadhighidentitywithmanyotherGPxs,especiallywiththoseofalgae,whichsharedthehighestidentityof66%withGriffithsiajaponica(Fig.3).TheconservedactiveaminoacidresiduesCys107,Gln141,andTrp197andGPxfamilysignaturemotifAFPCDQFandKWNFEKFLwerefoundintheGPxofG.lemaneiformis(Fig.1).
TheaminoacidsequencealignmentoftheputativeGRofG.lemaneiformisshowedhighidentityof59%withother
Results
CloningandanalysisofGPxandGRcDNAsequencesCompletecodingsequencesanddeducedaminoacidse-quencesofGPxareshowninFig.1.Thefull-lengthcDNA
Fig.3PhylogeneticanalysisofGPxaminoacidsequences.Numbersatthenodesrepresentthebootstrapvalues.Theevolutionarydistancebetweenthegroupsisindicatedbythescale.GPxsequencestakenfromGenBankwereasfollows:C.reinhardtii(EDP05620.1),Chlamydomonassp.W80(BAA83594.1),Volvoxcarterif.nagariensis(EFJ51396.1),Cyanothecesp.PCC8801(ACK67869.1),Cyanothecesp.PCC8802(ACV02776.1),A.thaliana(NP_180715.1),Glycinemax(ACU14676.1),Zeamays(AAM88847.2),O.sativajaponicagroup(BAD28380.1),Chlorellavariabilis(EFN60096.1),andG.ja-ponica(AAP80851.1)
JApplPhycol(2013)25:1925–1931Fig.4PhylogeneticanalysisofGRaminoacidsequences.Numbersatthenodesrepresentthebootstrapvalues.Theevolutionarydistancebe-tweenthegroupsisindicatedbythescale.GRsequencestakenfromGenBankwereasfollows:C.reinhardtii(EDP08556.1),ChlamydomonasspICE-L(ADI80343.1),V.carterif.nagariensis(EFJ53152.1),C.
speciesusingBLAST.ThephylogenetictreeindicatedthatGRofG.lemaneiformisisgroupedwithcyanobacteriumandisnotalignedwithotheralgaeandhigherplants(Fig.4).Theconservedregionsresponsiblefortheredox-activedisulfidebridge(GGTCVNVGCVPKK),theglutathione-bindingres-idues(TPVAIAEGandHPVSAEEMVT),andtheconservedresiduesforGSSGbinding(GGGYIA)arefoundintheaminoacidsequenceoftheputativeGRofG.lemaneiformis(Fig.2).
CloningofgenomicDNAofGPxandGRandgenomicSouthernblot
GPxDNAsequencesobtainedfromboththewildtypeandcultivar981wereofthesamelengthof750nucleotides,whichwasidenticaltotheORFinthecDNAencodingGPxsequence.GRDNAsequencesclonedfromthewildtypeandcultivar981werealsoidenticaltotheORFincDNAencodingGRsequence.TheseresultsindicatedthatbothGPxandGRgenesofG.lemaneiformiswerecontinuous,withoutintrons.Atthesametime,itrevealedthattherewerenodifferencesbetweenthewildtypeandcultivar981ofthesetwogenes.
TheSouthernblotresultsshowedthatbothGPx(Fig.5a)andGR(Fig.5b)weresingle-copygenesinboththewildtypeandcultivar981.TherewerenodifferencesinthegeneticcopiesofGPxandGRbetweenthetwodifferentstrainsofG.lemaneiformis.
ActivityassaysandrelativetranscriptiondetectionsofGPxandGRunderheatstress
TheactivitiesofGPx(Fig.6a)andGR(Fig.6b)weremeasuredinwildtypeandcultivar981grownunderhigh
1929
variabilis(EFN53075.1),A.thaliana(NP_191026.1),G.max(AAF26175.1),Z.mays(CAA06835.1),O.sativajaponicagroup(BAA11214.1),NodulariaspumigenaCCY9414(EAW45599.1),andCyanothecesp.PCC7424(ACK72699.1)
temperatures(32°C).TheinitialGPxactivityofthewildtypewasevenhigherthanthatofthecultivar981.Duringthestressphase,GPxactivityinthewildtypedeclinedandwaslowerthantheinitialactivityupuntil72hpost-stress.Inthecultivar981,afteradeclineat24h,GPxactivityincreaseddramaticallyduringthestressphase,upto199.5
Fig.5GenomicDNAdigestedwithendonucleasesandsouthernblottingofGPx(a)andGR(b)bothinthewildtypeandcultivar981.aW-1and981–1weredigestedwithKpnIandEcoRI;W-2and981–2weredigestedwithSacIandBamHI.bW-1and981–1weredigestedwithNotIandBamHI;W-2and981–2weredigestedwithNcoIandXhoI
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Fig.6ActivityassaysofGPx(a)andGR(b)bothinthewildtypeandcultivar981at32°C.Eachdatumrepresentsthemeanofthreedeterminationsonthesamesamplewithstandarddeviation
Umg−1proteinafter72h,whichwasovertwofoldhigherthantheactivityfoundinwildtype(97.4Umg−1protein).AssaysofGRactivityshowedthatwithorwithouthightemperaturestress,theGRactivityofcultivar981wasalwayshigherthanthatofthewildtype.Asthestresstimewasextended,thesuperiorityofcultivar981toadequatelyresistheatstressbecamemoreandmoreobvious.
ThemRNAtranscriptionsofGPx(Fig.7a)andGR(Fig.7b)werequantifiedafter0,6,12,24,48,and72hofheatexposureat32°C.Significantdecreasesinthetran-scriptionofGPxwereobservedoverthedurationofhigh-temperatureexposurecomparedwiththecontrolset(0h)inwildtype,whileincultivar981,nosignificantdecreaseinthetranscriptionofGPxwasobserved.TherewasonlyaslightdecreaseinGPxtranscriptionfrom6to12h,andthenaslightincreasewasobservedfrom12to48h.ThehighesttranscriptionlevelofGPxincultivar981wasobservedat48h.GRtranscriptionshowedsignificantdifferencebe-tweenthewildtypeandcultivar981,too.Incultivar981,GRtranscriptionwasupregulatedattheinitialtime(6h)andremainedatthehightranscriptionlevelforalongtime(till72h)underheatstress.Whileinwildtype,itwasdownregulatedduringthewholetimeofheatstress.
WithcomparisonoftheactivityandtranscriptionofGPxandGRinthetwostrainsofG.lemaneiformisunderheat
stress,higherlevelsandlongerdurationwerefoundintheheat-tolerantcultivar981.TheseresultsindicatethatthetranscriptionsofGPxandGRmaybedirectlyrelatedtotheheat-tolerantabilityofG.lemaneiformis.
Discussion
GPxandGRaretwoimportantantioxidantenzymes,whichplayimportantrolesinplantresponsestoenvironmentalstressorsincludingheatstress.GPxcancatalyzethereduc-tionofH2O2ororganichydroperoxidestoH2OoralcoholsbyGSH.GRcanreduceGSSGtoGSHandparticipateinprotectionagainstoxidationbymaintaininganadequateredoxstateintheintracellularenvironment.TheprocedureisthoughttobeanimportantreactionofthesystemforthedetoxificationofROSinplants.ThisstudyinitiallyreportsonthecloningofGPxandGRfromG.lemaneiformis.GPxisaproteincomprisedof249aminoacidswithaGPxfamilysignaturemotifAFPCDQFandKWNFEKFL.GRisapro-teincomprisedof471aminoacidswithconservedregionsresponsiblefortheredox-activedisulfidebridge(GGTCVNVGCVPKK),theglutathione-bindingsites(TPVAIAEGandHPVSAEEMVT)andtheGSSGbindingsite(GGGYIA).DNAsequencesofbothGPxandGR
Fig.7RelativetranscriptiondetectionsofGPx(a)andGR(b)bothinthewildtypeandcultivar981at32°C.Eachdatumrepresentsthemeanofthreedeterminationsonthesamesamplewithstandarddeviation
JApplPhycol(2013)25:1925–1931clonedfromthewildtypewereinconcordancewiththosefromcultivar981,andthesewereallsingle-copygenes.ThisrevealedthattherewerenomutationsineithertheGPxortheGRgenesbetweenthetwostrainsofG.lemaneiformis.
StudiesonIndianmustardcultivars(Gilletal.2011),Dioscorea(Zhangetal.2010)andmarinemacroalgae(Dummermuthetal.2003),havedemonstratedthatchangesinantioxidantenzymeactivitiesunderstresscanprovideinsightintoaplant'sabilitytotolerateheatstress.Thus,thebiologicalactivitiesandmRNAtranscriptionsofGPxandGRwereassayedforthetwostrainsofG.lemaneiformis.ItwasfoundthatincreasingordecreasingmRNAlevelschangedinaccordancewiththeactivityofGPxandGRintwostrainsofG.lemaneiformisexposedtohightempera-tures.BothGPxandGRwereupregulatedincultivar981anddownregulatedinthewildtypewhenexposedtoheatstress.ThisconfirmedthattheheightenedactivityandmRNAlevelsofGPxandGRincultivar981wererelatedtotheheattoleranceofG.lemaneiformis.
AlthoughtheGPxandGRsequenceswerehighlycon-servedinthetwostrainsofG.lemaneiformis,themRNAtranscriptionsandenzymaticactivitieswerequitedifferent.Promotersequence,transcriptionfactors,activityofRNApolymerase,activators,andrepressorscouldregulatethemRNAtranscriptions.Regulatorysequence,chaperons,andinhibitorscouldaffecttheproteintranslation.Moreover,otherheatshockfactors,suchasHSP,ubiquitin,etc.,wouldalsorespondtoheatshockandthenaffecttheactivityofGPxandGR.HowtheseelementsworktogethertodefendtheheatstressinG.lemaneiformisisnotclearnow.Furtherin-depthresearchisneededasasupplementtothisstudy.
AcknowledgmentsThisresearchwassupportedbytheResearchFundfortheNationalHighTechnologyResearchandDevelopmentProgramofChina(grantno.2012AA100811-3),theSpecialFundforAgro-scientificResearchinthePublicInterest(grantno.200903030),theNationalNaturalScienceFoundationofChina(grantno.30700608),andtheDoctoralProgramofHigherEducationofChina(grantno.20070423011).
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