1、TranscriptomeAssemblyUncoveredCandidateGenesInvolvedinPhytohormone 1*TranscriptomeAssemblyUncoveredCandidateGenesInvolvedinPhytohormone 1*1Collegeof Plant, 2 College, #Theseauthorscontributed equallytothe*Addresscorrespondence() erests:TheauthorsdeclarenoYellowhorn (Xanthoceras sorbifolium), the onl

2、y usXanthoceras, is a treetraditionally for fruits, oil productionand medical uses.With its profuse flowers, are now emerging as an ornamental plant in Norh Yellowhorn (Xanthoceras sorbifolium), the only usXanthoceras, is a treetraditionally for fruits, oil productionand medical uses.With its profus

3、e flowers, are now emerging as an ornamental plant in Norh America and Europe. However, e tedwithflowerandfruitdevelopmentinyellowhornisHere,wecarriedranscriptomeexperimenttoprofiletheexpressedgenesinflowerbuds,andfruitsofyellowhorn.Bydenovoysis,weidentified106,550unigenes.Next,alignedtheunigenestoN

4、RNTandUniprotdatabasesandprovidedtheir anno ions.Differential eOntologytermshose unigenes,phytohormonesignaltransductionandbiosynthesissofvarious entidentifiedregulatorygenesincytokinine,ethyleneandbrassinosteroidsignalingtransductionpathwaysup-regulatedinfruitscomparedwithflowerbudsandflowers;where

5、asesin patterns,which,mightbetoflowerandfruitdevelopment.Moreover,wealsoidentifiedalargenumberofSingle anoflowerandfruitdevelopmentinyellowhornandcouldserveasusefulinformationornamentalimprovementoffloweringplantsin tree.AstheonlyusXanthoceras,yellowhornis tree.AstheonlyusXanthoceras,yellowhornisnat

6、ivelygrowninnorthernChinahavetraditionallybeencultivatedforitsedibleleaves,flowers,fruitsandseeds.yellowhornfruitisanovalleatherycapsulecarries618seeds.Thematureseedsareabout1.5tcontainupto70%oil, n70%ofwhichareLinoleicacid(43%,C18:2)Oleicacid(30%,C18:1).The saturatedfattyacidsareonlycountsn7%ofyell

7、owhornYellowhornisalsofavoredforitswonderfulflowersandelegantshinygreenvebeenusedasasanornamentalplantinmericaandEurope.Yellowhornflowersare23diameter,withfivewhitepetals,fuseflowerscancoverthetreey.Moreover,theyellowhornfruitandseedsarerichsecondaryvel

8、ongbeenvedasahealth-lplant.Yellowhornmultiple dforthetreatmentofrheumarthritis,rheumaticernus,dermalrheumatismParkinsons素果實(shí)：auxin,CK,GA,ABA,ethyleneBRJAandSAHowever,theregulatorygenesinphytohormonesignalingpathwaysareremainedin transcriptomicstudies.Inparticular,素果實(shí)：auxin,CK,GA,ABA,ethyleneBRJAandSA

9、However,theregulatorygenesinphytohormonesignalingpathwaysareremainedin transcriptomicstudies.Inparticular,improvedalgorithmsarenowavailablefordereassemblingthetranscriptomeofanon-plantspecieswithoutavalidreference.Theassembledtranscriptscansubsequentlybeusedtoprofilegene network,andcharacterizegenet

10、icvariationsinRNAsequencing(RNA-seq)hasbeenwidelyanycasestodecipherdevelopmentalsesandvariousbiotic/abioticstressYellowhornarenowemergingasanimportant woodyplantforitsversatileusesinoil oftheflowerandfruitdevelopmentstillremainsunclearinhiserformed sequencingplatformwithpaired-endprotocal.Usingdenov

11、oysis,wetranscriptomeandanno edthembyaligningthetopublicdatabase.Next,weprofiledgenelevelsinflowerbuds,flowersandfruitsanduncoverede-expressedgenesusingdifferentialysis.Genefunctionandpathwaytthephytohormonesignaltransductionandsecondarymetabolitebiosynthesisthemostsignificantlyhe wealsoidentifiedal

12、argenumberofmolecularmarkersforfutureresearches.OurstudyprovidesvaluabledatasetforupdatingourunderstandingoferegulatorynetworkunderlyingflowerfruitdevelopmentinXanthoceras2.Materialand2.1.PlantMaterialsandGrowthTheseedsoftheXanthocerassorbifoliumwereprovidedbyAcademyof,China.Theseedsweregrowninsmall

13、pots 2.Materialand2.1.PlantMaterialsandGrowthTheseedsoftheXanthocerassorbifoliumwereprovidedbyAcademyof,China.Theseedsweregrowninsmallpots growthchamber(25C,12hforlight.And23C,12hfordark,).Thencultured seedlingstransferredto 2.3.RNAExtractionandcDNALibraryTotal RNA of les with three biological repli

14、cates was extracted according to MiniBESTUniversal RNAExtractionKit (TaKaRa) protocol. egrity ty of RNA les were checked using 2100 yzer with RNA 6000 Nano Assay Kit Technologies).les with egrityNumbers (RINs) nseven were and subjected to reverse-transcription for cDNAlibrary construction. The TrueS

15、eq RNA 2.4.IlluminaSequencingandThecDNAlibrarieswere sequenced using Illumina Hiseq 4000 platform(Illumina to generate 150 bp paired-end reads. We removed the RNA-seq reads containing adaptors contaminated reads, 5% sequencing errors and/or q-value n 5 to obtain reads.erformeddenovoassemblyusingTrin

16、ityprogram.Thelongestthe redundant andchimeric transcripts were consideredasunigenesand subjected to ionandcodingsequence(CDS) For coding sequence (CDS) The Unigenes were aligned to NCBI Non-Redundant Protein (NR), NCBI Nucleotide (NT) Swiss-Prot databases, eukaryotic Orthologous Groups For coding s

17、equence (CDS) The Unigenes were aligned to NCBI Non-Redundant Protein (NR), NCBI Nucleotide (NT) Swiss-Prot databases, eukaryotic Orthologous Groups (KOG), Gene Ontology (GO) and Encyclopedia of Genes and Genomes (KEGG) by BLAST with E-value cut-off of 1 10-5. best matchwas used to predicted the ive

18、 coding sequence region and determine the 5-We aligned the clean reads to the assembled unigenes using Bowtie. The read count number each gene was calculated using RNA-Seq by ization (RSEM). levels were normalized and estimated to reads per kilobase of transcript per fragments mapped (RPKM). DEseq2

19、was used to search for differentially expressed genes q-value n 0.005 and log2FoldChange n 1 n two les. GO ysis were subsequently performed for the DEGs GOseq based on the non-centralhypergeometricdistribution.KEGGysis of the DEGswascarried usingKEGGOrthologyBasedionSystem TwomicrogramsoftotalRNAfor

20、eachleweresubjectedtoDNAdigestionandtranscription to -strand cDNA using EasyScript One-Step gDNA Removal cDNA perMix Kit (Transgen Biotech, Beijing, China). The cDNAreaction wasdiluted 10-foldand2uLof the dilutionwasused tocarryoutreal-time PCRusingGreen qPCR SuperMix (Transgen Biotech) on a real-ti

21、me PCR system (LineGene 9600). real-time PCR program consists of 94 C for 5 min, 39 cycles of denaturing at 95 C for 10 annealing and at 59 C for 20 s. An actin gene was used as ernal control. relative level of detected genes fied by the 2-Ct algorithm Excel.Foreacheastthree biologicalreplicatestode

22、termine relativeannealing and at 59 C for 20 s. An actin gene was used as ernal control. relative level of detected genes fied by the 2-Ct algorithm Excel.Foreacheastthree biologicalreplicatestodetermine relativelevel.Therelatedprimersusedforreal-timePCRwereableRNA-SeqandDeNovoAssemblyoftheXanthocer

23、assorbifoliumWecarriedoutRNA-ysistoelucidateeregulationnetworkunderlyingfloweranddevelopmentinyellowhorn.Wegrowthyellowhornandisolatedflowerbuds,flowersandfruits1A).RNA paired-endprotocol.Afterfilteroutthelowqualityread(methods),weobtained318,119,840reads.Usingerformeddenovotranscriptomeassemblywith

24、thecleanreadsand127,325transcripts,fromwhich106,550unigeneswereobtained(Fig.1B).Thelengthofunigenesrangedfrom201bpto21,296withtheaveragemeanlength592ntandthelength1,046nt(Fig. Weusedbowtie2toalignthecleanreadtotheassembled.Of318,119,840cleanreads,299,549,945(94.2%)canbealignedto(Fig2A).Incases,RNA-s

25、eqexperimentmayproduceitionaltmayleadtoahighlevelassembly.WecheckedthedistributionofRNA-seqreadsonthetheassembledunigene.tthereadsweremainlyenrichedonthecentralregionofgenebody(Fig2B).ThesealsoconfirmedahighqualityofRNA-seq otal,identifiedORFencodingpeptidestleat100aminoacidfrom30,274unigenes(Fig.2C

26、).averagelengthofthecodingregionsis954.Usinge,BLASTPandBLASTX,wesearchedtheanno ionfortheunigenesbyaligningtheassembledtranscriptsandpredictedpeptidetoNCBINon-RedundantProtein(NR),NCBI(NT)andSwiss-Prototal,weidentified44,194unigenessignificantmatchestotheedgenes/proteinseastonedatabase(Fig.2D).tocon

27、servative,wealsotoNCBINon-RedundantProtein(NR),NCBI(NT)andSwiss-Prototal,weidentified44,194unigenessignificantmatchestotheedgenes/proteinseastonedatabase(Fig.2D).toconservative,wealsoalignedtheunigenesto GOandKEGGdatabases.TheresultswereshowinSupplementalTableX,whichcouldasareferenceionforthefutureu

28、p entof function”and”cellularcomponent”asthe3majorclasses. Inallthecomparisons,GOtermsWemeasuredlevelsofunigenesbycalculatingnormalizedReadsPerKilobaseMappedReads(RPKM).TheunigenewithnKMereusedperformhierarchicalysisbasedonthePearsoncorrelationefficiencyofthentwoles.Thehierarchicaltreetthebiological

29、replicateswereclusteredtogetherindicatingahighlyreproducibilityofourdetectionnreplicates(Fig.Next we searched for the differentially expressed unigenes betn different tiesusing DESeq2. Ourysis identified 23,573 differentially expressed unigenes(foldchange oflevel betn ties 2 and P-values 0.05 , Fig.

30、 3B). The most clear transcriptome changewasobservedinopenflowerscomparedwithflower budsandfruits(SupplemtnaltableX). There were 12,506 differentially expressed unigenes co-occurredhe 2 comparisons; whereas those in flower buds and fruits were 2848 and 3509, respectively. The differentially expresse

31、dnopenflowersandflowerbuds,nfruitsandopenflowersaswellas nandflowerbudswereprovidedinsupplementaltablex- s”showedmoreabundantent ).Ofthem,theGOterm“hormone- s”showedmoreabundantent ).Ofthem,theGOterm“hormone- entsthe ans.Wealsodetectedthehighlyentsoffruitdevelopment”(GO:0010154,P-value1e20)and“secon

32、darymetabolic Tofurtherdissecttheregulationpathwaysof KEGGysis.Unexpectedly,wefoundmostofthehighlyenrichedpathways table).Table1listedthetop20mostsignificantlyoverrepresentedpathwaysoffruit-expressedunigenes.Theresultclearlyttheseunigeneswereenrichedinplant acid,nicotine,carotenoid,cutin,flavonoidan

33、dysisidentifiedtheregulatorygenesinauxin,CK,GA,ABA,ethylene,BR,JAand unigenesinplanthormonesignaltransductionpathwayasanletopresentthenflowersandfruitsinyellowhorn(Fig.4A).CRE1isthecytokinine(CK) AHPregulatesthefeedbackloopofthetranscriptionfactorARRbandtmodulatetranscriptionofdownstreamgenes.Inourd

34、atasets,weobservedtheup-regulatedofXsAHP,XsARRaandXsARRbinCKsignalingtransductions.Inplants,BRI1andBAK1perceivesbrassinosteroidandphosphorylatesBSKandBKI1,whichsubsequentlytransferssignaltoBZR1/2throughBSU1,BIN2.TheactivatedformofBZR1/2promotesthegeneCYCD3andTCH4tocontrolcellandenlongation.Inourstud

35、y,wefoundXsBKI1andXsCYCD3werepreferentiallyexpressedinfruits.Inethylenesignalingpathway,ETHYLENERESPONSE1(ETR1)canformhomo-dimertobindethyleneanderactwithCONSTITUTIVETRIPLERESPONSE1(CTR1).Then,thesignalscansequentiallytransducedtoSIMKK,MPKXsBKI1andXsCYCD3werepreferentiallyexpressedinfruits.Inethylen

36、esignalingpathway,ETHYLENERESPONSE1(ETR1)canformhomo-dimertobindethyleneanderactwithCONSTITUTIVETRIPLERESPONSE1(CTR1).Then,thesignalscansequentiallytransducedtoSIMKK,MPKandEIN2andfinallytoactiveEIN3andERFpromotedownstreamgenes.EIN3canbedegradedbyEBF1throughubiquitin- ysisshowedthelevelsofXsETR,XsEIN3,XsERF1andwereup-regulatedinfruitscomparedwithheans.Differentfromes inBRandethylenesignaling pat