兩個溫度下Picrohiza kurrooa轉(zhuǎn)錄組的從頭組裝和特征描述顯示轉(zhuǎn)錄組的調(diào)整

兩個溫度下Picrohizakurrooa轉(zhuǎn)錄組的從頭合成和特征描述顯示轉(zhuǎn)錄組的調(diào)整背景PicrorhizakurrooaRoyleexBenth.isamedicinallyimportantendangeredplantspeciesoffamilyScrophulariaceae.Thespeciesisdistributedbetween3000-5000mabovemeansealevelintheHimalayanregion.P.kurrooaiswidelyusedintraditionalaswellasmodernsystemofmedicineforthetreatmentofliverdisorders,fever,asthmaandjaundice.Indiscriminateandextensiveharvestingandlackoforganizedcultivationhasthreatenedthestatusofthisplantinnatureandislistedas“endangeredspecies”byInternationalUnionforConservationofNatureandNaturalResources.胡黃連提取物來自有藥用價值瀕危的玄參科植物物種。此物種分布在高于平均海平線3000-5000m的喜馬拉雅地區(qū)。P.kurrooa廣泛用于傳統(tǒng)和現(xiàn)代醫(yī)藥系統(tǒng)，用于治療肝疾病、發(fā)燒、哮喘和黃疸。任意大量收割和缺乏組織培養(yǎng)威脅著它在自然界中的地位，并被國際自然保護聯(lián)盟和自然資源學(xué)院列為瀕危物種。ThebiologicalactivityofP.kurrooaisattributedtothepresenceofiridoidglycosidesmainlypicrosideIandpicrosideII.Studieshaveshownthattemperatureplaysanimportantroleinthebiosynthesisandaccumulationofpicrosides.Atemperatureof15℃favoredpicrosidesaccumulationascomparedto25℃andthiswasinagreementwiththeexpressionof1-deoxy-D-xylulose-5-phosphatesynthase(DXS)and3-hydroxy-3-methylglutaryl-coenzymeAreductase(HMGR),thegenesassociatedwithpicrosidesbiosynthesis.However,theprogressinunravelingthemolecularresponseofP.kurrooaatthesetemperatureshasbeenimpededbythedearthoftranscriptomicresources.胡黃連的生物活性歸功于環(huán)烯醚萜苷的存在，主要是胡黃連苦苷I和II。研究表明，溫度在胡黃連苦苷的生物合成與積累中有重要作用。與25℃相比，15℃有利于胡黃連苦苷的積累，這與1-脫氧-d-木酮糖-5-磷酸合成酶(DXS)和3-羥基-3-甲基戊二酰-輔酶A還原酶(HMGR)的表達一致，這些基因與胡黃連苦苷的生物合成相關(guān)。However,theprogressinunravelingthemolecularresponseofP.kurrooaatthesetemperatureshasbeenimpededbythedearthoftranscriptomicresources.Transcriptomesequencingisanefficientwaytounderstandglobalmolecularresponsebytheplantinresponsetoacue.Transcriptomesequencingthroughnextgenerationsequencingtechnologyprovidesextensivedatainmuchshortertimeperiodwithenormousdepthandcoveragetofacilitateunderstandingofmajorchangeinthemetabolicprocessesaswellascontributetocomparativetranscriptomics,evolutionarygenomicsandgenediscovery.然而，揭開P.kurrooa對溫度的分子反映過程受到缺乏轉(zhuǎn)錄組資源的阻礙。轉(zhuǎn)錄組測序是通過植物了解全球分子反應(yīng)線索的有效方法。通過第二代測序技術(shù)的轉(zhuǎn)錄組測序在短時間內(nèi)提供大量數(shù)據(jù)，用龐大的深度和覆蓋度來促進理解生物合成過程中的主要變化，也有助于比較轉(zhuǎn)錄組學(xué)、進化基因組學(xué)和基因的發(fā)現(xiàn)。ThepresentstudydescribesthefirstglobalanalysisofP.kurrooatranscriptomeundertwotemperatureregimes,whichwouldserveasablueprintofgeneexpressionprofile.Theworkreportsastrategyfordenovoassemblyoftranscriptomeusingshort-readsequencedatageneratedbyIlluminaRNA-Seqmethod.Readperexonkilobasepermillion(RPKM)basedcomparativeexpressionprofilingstudywasdonetosystematicallycharacterizethemRNAsattwotemperaturesandtoidentifythedifferentiallyregulatedgenesincludingthoseinvolvedinpicrosidesbiosynthesis.Dataoninsilicogeneexpressionwasvalidatedbyreversetranscriptase-polymerasechainreaction(RT-PCR)aswellusingasetof19genes.目前研究第一次描述了在兩個溫度體系下P.kurrooa轉(zhuǎn)錄組的全面分析，這將作為基因表達譜的一個藍圖。這個工作報道了轉(zhuǎn)錄組從頭合成裝配IlluminaRNA測序方法生成的短讀數(shù)序列數(shù)據(jù)的戰(zhàn)略。根據(jù)比較表達譜研究Readperexonkilobasepermillion(RPKM)用來系統(tǒng)地描述在兩個溫度下的mRNA，并鑒定包括那些涉及胡黃連苦苷生物合成在內(nèi)的不同調(diào)控基因。計算機基因表達數(shù)據(jù)由19個基因的逆轉(zhuǎn)錄酶鏈式反應(yīng)確認。方法植物材料P.kurrooa收集于天然生境，轉(zhuǎn)移到植物生長室用16h光周期維持在15℃和25℃。充分澆水并在第六天對兩個溫度抽樣。收獲第三葉片用于各種試驗，液氮凍存存于-80℃。提取和估計胡黃連苦苷高效液相色譜儀系統(tǒng)，檢測胡黃連苦苷并使用胡黃連苦苷I和II作為標準進行量化。使用四個重復(fù)。方法準備cDNA及轉(zhuǎn)錄組測序

總RNA提取、純化。Illumina平臺測序。從頭組裝和序列集群

k-mer大小為23時獲得重疊群數(shù)量、覆蓋度和平均序列長度之間的最好平衡。組裝驗證和組裝transcripts相似性搜索

為了評價組裝的真實性，使用在dbESTNCBI可以得到的500個實驗室P.kurrooaEST序列。通過BLASTX針對NR蛋白數(shù)據(jù)庫用E值界限10-5進行掃描。序列注釋

用E-值界限10-1，對UniProt數(shù)據(jù)庫blast，結(jié)合GO、KEGG、EC。在15°C和25°C，為序列執(zhí)行這種富集分析評估各種GO分類有2倍或以上的表達水平。功能域搜索未知序列

通過BLASTX沒有返回任何同源序列匹配的組裝序列，被轉(zhuǎn)換成六個最長的ORFs，它使用RPS-BLAST瀏覽像保守結(jié)構(gòu)域數(shù)據(jù)庫那樣的功能域數(shù)據(jù)庫。為不同環(huán)境條件搜索比較相似序列組裝

為找到15°C和25°C組裝序列之間共同轉(zhuǎn)錄組序列，兩種種序列使用BLASTN用E值閾值10-5互相搜索。最佳匹配確定為共同轉(zhuǎn)錄序列。read映射和轉(zhuǎn)錄組序列豐富度測量

對于RPKM測量，首先映射過濾的reads到各種組裝序列，估計分配給每個組裝轉(zhuǎn)錄組序列的總映射reads、獨特的映射reads，最多允許兩個不匹配。SeqMap用于read映射，而rSeq應(yīng)用于基于表達測量的RPKM。為每個序列收集來自兩樣本的表達數(shù)據(jù)。查找樣本間相似序列，通過計算在15°C和25°C表達比率測量它們的差異表達。GC含量分析和SSRs的鑒定

用EmbossGeeCee工具測量GC含量，用MISA掃描序列尋找SSR標記。基因驗證和表達分析

基因表達的結(jié)果由RT-PCR驗證。每個反應(yīng)執(zhí)行三次，26srRNA為內(nèi)部參考標準。結(jié)果與討論ThepresentworkwascarriedoutondenovotranscriptomesequencingofP.kurrooa,denovoassemblyofshortreads,annotationofassembledsequencesat15°Cand25°CandvalidationofRPKMbasedexpressionanalysisbyRT-PCRusingselectedgenes.P.kurrooaisamedicinallyimportantandendangeredplantspecies.Medicinalpropertiesareattributedtothemonoterpenoids,picrosideIandpicrosideII,whichareassociatedwithhepatoprotectiveactivityasoneofthemajoractivities.目前工作是進行從頭合成15℃和25℃的P.kurrooa轉(zhuǎn)錄組、短序列裝配、拼接序列的注釋,以及使用基于表達分析選擇基因進行RT-PCR進行RPKM確認。P.kurrooa是一個瀕臨滅絕的重要藥用植物，藥用性質(zhì)歸功于單萜、胡黃連苦苷I和II，它們與主要功能保肝藥活性相關(guān)。reads生成和從頭合成序列組裝Paired-end(PE)runof36cycles,foreachoftheleaftissuescollectedfromplantsexposedto15°Cand25°C,wasperformedonIlluminagenomeanalyzerIIxplatform.BclconverterwasusedtoproducethereadsinqseqformatofthePErunofgenomeanalyzer.Itcontainsreads,theircoordinates,tilenumberandqualityencoding.Since3’endsofreadsarepronetosequencingerror,forevery36bpread,only33bases(excludingthe3basesat3’end)wereconsideredforfurtheruse.Atotalof27,562,496and49,274,224PEreadsweregeneratedat15°Cand25°C,respectively.Afterperformingqualityfiltering,atotalof20,593,412and44,229,272PEreadswereobtainedfor15°Cand25°C,respectively.Intotal64,822,684PEreadswereobtained.雙末端運行36循環(huán)，從15℃和25℃條件下分別收集葉片組織，執(zhí)行illumina基因組IIx分析平臺。Bcl轉(zhuǎn)換器用于生產(chǎn)分析基因組的qseq格式的讀數(shù)，包括reads、它們的坐標、組號、編碼質(zhì)量。由于3’末端讀書容易出錯，每36bpread只有33個堿基（排除3’末端的3個堿基）后續(xù)使用。15℃和25℃分別有27,562,496和49,274,224reads。質(zhì)量篩選之后，15℃和25℃分別有20,593,412和44,229,272reads，總共得到64,822,684reads。Inordertoselectthemostappropriatek-mersizeforconsideringdenovoassembly,SOAPdenovowasrunatdifferentk-mersizerangingbetween19to29mers,withreadlengthof33bp.Theparametersrecordedwerethetotaltranscriptsobtainedafterassembling,averagecoverage,averagetranscriptsize,percentageoftranscriptshavinglengthhigherthan1,000bpandhighesttranscriptlength.K-mersizeof23meremergedasthebestchoiceforperformingassembly,asithadabalancebetweenover-representedandunder-representedtranscriptnumbers,coverage,maximumlengthobtainedandaveragetranscriptlength(Table1).為了選擇從頭合成組裝的最合適k-mer大小，在19到29mers的范圍使用不同的k-mer大小運行SOAP從頭合成，使用33bp長度的reads。記載從組裝獲得的全部轉(zhuǎn)錄組的參數(shù)，平均覆蓋度、平均轉(zhuǎn)錄大小、長度大于1,000bp的百分比、最大長度。23mer產(chǎn)生的K-mer大小作為最佳方法執(zhí)行組裝，因為在轉(zhuǎn)錄組表達上調(diào)與下調(diào)數(shù)量、覆蓋、最大長度和平均轉(zhuǎn)錄長度之間有一個平衡（表1）。Thedatafromthetwotemperatureconditionswereassembledseparately.For15°Cassembly,atotalof31,338assembledtranscriptswithaveragelengthof403.87bpandaveragecoverageof64.68timeswasobtained(Table2).Total2,029assembledtranscripts(6.48%)hadsequencelengthlongerthan1,000bpwiththelongestassembledtranscriptof5,326bp.Similarlyfor25°C,63,718assembledtranscriptsequenceswereobtainedwith434.39bpaveragelengthandaveragecoverageof71.26.Total4,988sequences(7.82%)werelongerthan1,000bpwithmaximumsequencelengthof5,210bp.兩個溫度的數(shù)據(jù)分別組裝。15℃組裝，總共31,338組裝序列，平均長度403.87bp,平均覆蓋64.68倍（表2）?？偣?，029組裝序列（6.48%）序列長度大于1000bp，最長拼接序列5,326bp。25℃，獲得63,718個組裝轉(zhuǎn)錄序列，平均長度434.39bp，平均覆蓋71.26次?？偣?,988序列（7.82%）長度大于1000bp，最大序列長度5,210bp。表2顯示在

15°C、25°C的組裝數(shù)據(jù)，最好的重疊群在混合樣本中，覆蓋率和平均長度都比前兩者好。Consideringthequalityofassembliesproducedattwodifferenttemperatures,itappearedthatincreasedreaddatacouldenhancethetotalcoverage,averagecontiglengthandpercentageoftranscriptslongerthan1kb.Therefore,PEreadsofthetwolanes(15℃and25℃)werecombined,retainingthePEinformation.Combineddenovoassemblywascarriedoutwith64,822,684filteredreadsinPEform(Table2).Withthisapproach,atotalof74,336transcriptswereobtained,ofwhich9.16%wereabove1,000bp,yieldinghigheraveragecoverageof76.6,averagelengthof439.5bpandmaximumlengthof5,759bp.Therefore,total74,336transcripts,generatedfrompooledreads,madethefinalrepresentativesforassembledsequencesinthisstudy.考慮到兩個溫度的組裝產(chǎn)物質(zhì)量，似乎混合的read數(shù)據(jù)總覆蓋、平均重疊群長度和大于1000bp序列百分比都較好。因此，15°C和25°C的PEreads結(jié)合到一起，64,822,684過濾reads從頭合成組裝（表2）。用這種方法，總共得到74336序列，其中9.16%大于1000bp，平均覆蓋76.6%，平均長度439.5bp，最大長度5759bp。因此，從reads池中總共獲得序列作為本研究中組裝序列的最終代表。同源性搜索和序列聚類Setofassembledsequencescontainedseveralsequencessharingsimilarity,causingover-representationofdatafortotaltranscriptsequencemeasurement.Suchredundancyandover-representationcanbereducedbyfindingsimilarsequenceseitherthroughmergingthemorebyusingasinglerepresentativesequenceinstead.Thiswasdonebyapplyingsequencesimilaritybasedclustering.AfterperforminghierarchicalclusteringwithTIGRGeneIndicesclusteringtools(TGICL)usingContigAssemblyProgram(CAP3)andClusterdatabaseathighidentitywithtolerance(CD-HIT),withminimum90%similaritycutoff,thenumberofuniqueassembledtranscriptsreducedfrom74,336to72,220.一套組裝序列包含幾個共享相似序列，導(dǎo)致總的序列測量數(shù)據(jù)高表達，通過尋找相似序列，合并它們或者使用單個序列作為代表能減少這中冗余和高表達，這些通過應(yīng)用相似序列聚類完成。在用TIGR基因指數(shù)聚類工具(TGICL)執(zhí)行層次聚類后使用CAP3和CD-HIT，用最小90%的相似度去除，獨特的組裝序列從74,336減少到72,220。Analysisofsequences,obtainedafterclustering,wasdoneforhomologysearchagainstproteinsequencesatnon-redundant(NR)databasesatNCBIbyBLASTXwithcutoffE-valueof10-5.Forpooledreadassembledtranscriptsequences,significantBLASThitswerefoundforatotalof42598sequenceswhilenohitwasfoundfor29,622sequences.AnotherclusteringstepwascarriedoutforsequenceswhichreturnedsignificantBLASThits.Sequenceswithnoapparentsignificantidentityamongthemselvesmightbelongtothedifferentpartsofthesamegeneormayrepresenttheisoforms.Countingthemasseparatetranscriptswouldonlyinflatethenumberofuniquegenes.獲得聚類后序列分析，在NCBI非冗余蛋白序列（NR）數(shù)據(jù)庫通過BLASTX查找同源性，刪掉E-valueof10-5?；旌铣豶ead組裝序列，有意義的BLAST匹配發(fā)現(xiàn)總共42,598序列，29,622序列沒有匹配。為序列實施另一種聚類步驟，它獲得有意義的BLAST匹配。序列之間沒有明顯的顯著特性，可能屬于同一基因的不同部分或者代表亞型。把它們作為單獨的轉(zhuǎn)錄組序列可能增大獨特基因的數(shù)量。Therefore,allthosetranscriptsweresearchedthatexhibitedsignificanthitsandsharedthebesthitstothesamereferencesequence.Asetoflocalscriptswaswrittentoscanforallthoseassembledtranscriptsequencesthatreturnedacommonbesthitandcommonreferencegenebutdifferedintheirlocation.Allsuchtranscriptswereclusteredtogetherandassumedasthemembersofassociatedreferencesequence/generepresented.ThisstepreducedthetotalnumberoftranscriptswithsignificantBLASThits,from42,598to28,403.因此，搜索所有這些存在顯著匹配并與同一個參考序列共享最優(yōu)匹配的轉(zhuǎn)錄序列。一個本地程序用于掃描這些組裝轉(zhuǎn)錄序列，它們返回一個共同最佳匹配并且共同參考基因但是所在位置不同。所有這些轉(zhuǎn)錄序列聚集在一起，并假設(shè)是相關(guān)參考序列/基因表達的成員。這一步用顯著BLAST匹配將轉(zhuǎn)錄序列總數(shù)從42,598減少到28,403。對比P.kurrooaESTs驗證組裝序列TheassembledsequencesobtainedfrompooledreadswerevalidatedbysequencebasedalignmentsagainstESTsofP.kurrooasubmittedatNCBIdbESTbyourgroup.For500submittedESTs,BLASTNanalysisagainsttheassembledtranscriptswasperformedwithanE-valuethresholdof10-05.Also,mostoftheassembledtranscriptsequenceswerefoundtobealignedcorrectlyandincontinuousform,withaverageidentityof96.35%,suggestinggoodassemblyquality.用我們的群組通過序列堿基與NCBI上dbESTP.kurrooaESTs比對來驗證從混合池reads中獲得的組裝序列。對于遞交的500個ESTs，BLASTN分析向組裝transcripts，執(zhí)行E-value臨界值10-05。而且，發(fā)現(xiàn)大部分組裝transcript序列被正確比對且為不連續(xù)的形式，平均一致96.35%，說明組裝質(zhì)量很好。使用轉(zhuǎn)錄組數(shù)據(jù)分析GC含量并鑒定SSRsAssembledtranscriptsequencesofP.kurrooawereanalyzedfortheseSSRs.Atotalof1,562SSRswereidentifiedintheassembledtranscripts.Thetri-nucleotideSSRshavebeenobservedtobethemostprevalentgroupofmarkers(45.63%)withhighestoccurrenceofGAA,TGG,CCA,AGA,andTCArepeatsfollowedbymononucleotide(35.25%)anddinucleotide(21.29%)SSRs.P.kurrooa轉(zhuǎn)錄組組裝序列分析用于SSRs。共鑒定出1562個SSR。其中以GAA、TGG、CCA、AGA、TCA重復(fù)為主的3-核苷酸重復(fù)最為普遍（45.63%），其次是單核苷酸重復(fù)（35.25%）和二核甘酸重復(fù)（21.29%）。最普遍的單核苷酸重復(fù)是poly-A，最普遍的二核苷酸重復(fù)是poly-AG和poly-TC。觀測到的poly-A可能由于RNA序列的poly-A。只有小部分的4-、5-核苷酸重復(fù)。一般來說，與2-、4-相比，3-核苷酸重復(fù)是最常見的。P.kurrooa轉(zhuǎn)錄組的功能注釋和分類ForfunctionalannotationofP.kurrooatranscriptome,transcriptswerecomparedagainsttheNRproteinsequencesavailableatUniProtdatabaseusingBLASTXalgorithmwithE-valuethresholdof10-01.TheassociatedhitsweresearchedfortheirrespectiveGeneOntology(GO),KyotoEncyclopediaofGenesandGenomes(KEGG)andEnzymeCommissionCodes(EC)foreachquerysequenceandthehighestbitscorehitwasselected.GOtermsarederivedfromdynamiccontrolledvocabulariesorontologiesthatcanbeusedtodescribethefunctionofgenesandgeneproducts.對于P.kurrooa轉(zhuǎn)錄組功能注釋，用BLASTN算法將組裝序列與UniProt數(shù)據(jù)庫的NR蛋白序列比對，E-value閾值為10-01。相關(guān)匹配用于尋找每個查詢序列它們各自的GO、KEGG和EC，并選擇最高得分。AnnotationagainstGOdatabaseyieldedsignificantannotationfor31,959outof72,220assembledsequences,representingthebestpossiblehits.Thesegeneswerefurtherclassifiedintotwomajorcategoriesnamely,biologicalprocessandmolecularfunctionusingplantspecificGOslimsthatprovideabroadoverviewoftheontologycontent.FunctionalclassificationofP.kurrooatranscriptsinbiologicalprocesscategory(Figure1A)showedthatmetabolicprocess,transport,regulationofbiologicalprocesses,responsetostimulusandcellularprocesswereamongthehighlyrepresentedgroupsindicatingthattheplantisundergoingrapidgrowthandextensivemetabolicactivity.GenesinvolvedinDNAbinding,catalyticandtransferaseactivitywerehighlyrepresentedinmolecularfunctioncategory(Figure1B)indicatingdominanceofgeneregulation,signaltransductionandenzymaticallyactiveprocesses.72290組裝序列中31959條得到有效的GO注釋，代表最可能的匹配。使用植物特殊GO注釋，將這些基因分為生物過程和分子功能兩個主要類別。P.kurrooa轉(zhuǎn)錄組序列在生物過程類別的功能分類（圖1A）代表有種群之間的代謝過程、轉(zhuǎn)運、調(diào)節(jié)生物過程，回應(yīng)刺激和細胞過程，表明植物正經(jīng)歷快速增長和大量代謝活動；分子功能分類的代表有DNA結(jié)合、催化和轉(zhuǎn)移酶活動性（圖1B）,表明主導(dǎo)基因調(diào)控、信號轉(zhuǎn)導(dǎo)和酶活性過程。圖1A

生物過程與A.thaliana和

M.truncatula相比P.kurrooa轉(zhuǎn)錄組的GO分類。兩個主要的GO分類：胡黃連擬南芥蒺藜苜蓿圖1B

分子功能AcomparisonfordistributionoftranscriptsacrossvariousGOslimcategories,betweenA.thaliana,P.kurrooaandM.truncatulashowednomajordifferencesbetweentheranksofGOslimcategories(Figure1A,B).However,comparisonfordistributionoftopoccurringGOtermsinsteadofGOslimcategoriesbetweenP.kurrooaandA.thalianasuggesteddifferentialdistributionoftranscriptsbetweenvariousGOcategories(Additionalfile8).通過GOslim分類比較A.thaliana,P.kurrooa和M.truncatula之間轉(zhuǎn)錄組序列的分布表明GOslim分類行列沒有顯著的差異（圖1A,B）。而用topoccurringGOterms代替GOslim分類時，P.kurrooa和A.thaliana顯示轉(zhuǎn)錄組序列多種GO分類差異分布。BestECclassificationwasobtainedfor14,630assembledsequences,whereasassociatedKEGGclassificationwasobtainedfor15560assembledsequences.Figure2Aliststhetop50abundantenzymeclassesobservedforP.kurrooatranscriptome.Interestingly,alargeamountofassembledtranscriptsbelongedtoserine/threonineproteinkinaseenzymeclassalone(14.6%).Besidesthis,Figure2Bdisplaystop50KEGGpathwaysrepresentedbytheassembledtranscriptomesequences.Highestnumberofsequencesbelongedtoplantpathogeninteractionpathways(6.13%).Highestrepresentedgroupsincludedmanypathwaysassociatedwithhousekeepingprocessesaswellasplantdevelopmentandsecondarymetabolism.為14630組裝序列獲得最好的EC分類，15560組裝序列獲得KEGG分類。圖2A轉(zhuǎn)錄組的50豐富的酶類。大量組裝序列只屬于絲氨酸/蘇氨酸蛋白質(zhì)激酶酶類(14.6%)。圖2B展示組裝序列代表的的前50個KEGG通路。大量序列屬于植物-病原相互作用通路（6.13%）。最高代表性種群包括許多結(jié)合管家過程和植物發(fā)展次級代謝的通路。在兩個溫度下的轉(zhuǎn)錄組分析顯示植物主要過程的調(diào)整Oneofthegoalsoftranscriptomesequencingwastocom-paretranscriptsat15°Cand25°C,thetemperatureswhichmodulatepicrosidescontent.Consideringdissimilarsequenceclusteringtocontainover-representation,GOannotationfor19,769uniquegeneswasobtainedalongwiththeirRPKMvaluesatthetwotemperatures.Basedonfoldincrementat15°C,thesegenesweregroupedintosixdifferentexpressionclassesrangingfrom>10foldincrementto0.5foldorlower.Highestnumberofgenesfellintothegroupwithminimumchangeinexpression.轉(zhuǎn)錄組測序的一個目的是比較在15℃和25℃，胡黃連苦苷的含量調(diào)整。我們的數(shù)據(jù)與先前工作顯示的15℃比25℃的胡黃連苦苷含量高一致（圖3）。考慮到不同序列聚集成表達上調(diào)，用RPKM值在兩個溫度下，為19769獨特基因獲得GO注釋。根據(jù)15℃倍增，把這些基因分為6組不同表達分類范圍從>10倍增長到0.5倍或更低。大量基因表達變化最小而落入這個范圍。圖3Molecularfunctiongroupsrepresentingmonooxygenaseactivity,2-iron,2-sul-furclusterbinding,cobalaminbinding,betalactamaseactivity,aminobutyraldehydedehydrogenaseactivity,purinetransmembranetransporteractivityandmetalionslikeironandcopperbindingactivitieswerefoundtobeover-expressedat15℃.Underthebiologicalprocessgroups,thoseassociatedwithvariousbiosynthesisandtransportprocessessuchaszincandammoniumtransportandproteinchromophorelinkagewereprominentat15°C.Figure4and5representthetop10biologicalprocessandmolecularfunctioncategories,respectively,presentattwofoldorhigherexpressiongroupsat15°Cand25°C.分子功能組：單氧酶活性、2-鐵，2-硫結(jié)合群、鈷胺素結(jié)合、β-內(nèi)酰胺酶活性、氨基丁醛脫氧酶活性、嘌呤橫跨膜的運輸活動、金屬離子如鐵、銅結(jié)合活性，在15℃高表達。生物過程組：那些與各種生物合成和運輸過程，如鋅和銨運輸和蛋白質(zhì)發(fā)色團連鎖有關(guān)的過程在15℃突出。圖4、5分別代表在15℃和25℃出現(xiàn)在2倍或更高表達的前10個生物過程和分析功能分類。圖4圖5Ascanbeseenfromthesediagrams,responsetoheat,responsetobioticstimulusandlipidcatabolicprocesswereexclusivelyrepresentedat25°C.Whileat15°C,categoriesforredox,glycogenbiosyntheticprocess,biosyntheticprocessesandproteinchromophorelinkagewereexclusivelyrepresented.Underthemolecularfunctioncategories,monooxygenaseactiviies,peptiaseactivities,catalyticactivities,2-sulfur,2-ironclusterbindingandproteinbindingactivitieswerefoundtobemorepronouncedat15°C,whilepectinesteraseandproteinkinaseactivitieswerepredominantat25°C.Afurtherenrichmentanalysisforfunctionacategoriespointedoutthattranscriptsassociatedwithresponsetostress,responsetostimulus,phytosteroidmetabolicprocessandbrassinosteroid(BR)metabolicprocessweresignificantlyenrichedinthegrouphavingtwofoldorhigherexpressionat25°C(Figure6).從這些圖可以看到,對熱反應(yīng),生物刺激反應(yīng)和脂質(zhì)降解過程只在25℃。而在15℃,專有表現(xiàn)氧化還原反應(yīng)、糖原生物合成的過程、生物合成過程和蛋白質(zhì)發(fā)色團連鎖。在分子功能分類中，單氧酶活性、肽酶活性、2-硫、2-鐵集群結(jié)合和蛋白質(zhì)結(jié)合活性在15℃更明顯,而果膠酯酶和蛋白激酶活性最主要在25℃顯著。對功能分類進一步富集分析指出，相關(guān)壓力反應(yīng)、刺激反應(yīng)、植物甾醇代謝過程和油菜素類固醇（BR）代謝過程顯著豐富，在25℃群組中有2倍或更高的表達（圖6）。Comparedtotheexpressionat15°C,up-regulationofstressresponsivetranscriptsissuggestiveofP.kurrooatobeunderstressat25°C.Theabovedescribedglobalanalysisofgeneexpressionprovidedcomprehensivedatasetwitheachgenerepresentedbyitsabsoluteexpressionlevelatthetwotemperatures.Severalprocessessuchasmetabolicprocesses,cellularprocesses,transportprocesses(Figure4),andcatalyticprocesses(Figure5)wereequallyrepresentedatboththetemperaturesindicatingtheimportanceoftheseprocessesinplantgrowthandsurvival;althoughdifferentsetofgenesintheseprocessesmightdeterminetheresponseofplanttotemperaturechange.Transcriptsassociatedwiththeprocessesinvolvinglipidmetabolismwerehighlyenrichedat25°C(Figure4),suggestingachangeinlipidprofile.Indeed,temperaturehasbeenshowntomodulatelipidprofileinplant.與15℃的表達相比，壓力反應(yīng)轉(zhuǎn)錄序列上調(diào)暗示了P.kurrooato在25℃時處于壓力之中。一些過程,如代謝過程、細胞過程、運輸過程(圖4)和催化過程(圖5)在兩個溫度中同樣表現(xiàn)，暗示了這些過程對植物生長和生存的重要性;盡管在這些過程中不同的基因隨溫度變化。涉及脂質(zhì)代謝過程的轉(zhuǎn)錄序列是在25°C高度富集(圖4),說明脂質(zhì)變化。實際上,已證明溫度調(diào)節(jié)植物脂質(zhì)。與胡黃連苦苷生物合成相關(guān)的通路顯示溫度依賴的調(diào)整Picrosidesareterpenoidswithaniridoidskeletonofmonoterpeneorigin.Dependinguponthefunctional-groupmoieties,picrosidesareclassifiedaspicrosideIandpicrosideII.PicrosideIhascinnamatemoiety,whereaspicrosideIIhasvanillatemoiety.ThecinnamateandvanillatemoietiesarederivedfromPPpathway(Figure7).Synthesisofcinnamaterequirestheactionofphenylalanineammonialyase(PAL)onphenylalaninewhereascaffeoyl-CoA3-Omethyltransferase(COMT)isthekeyenzymeforvanillatebiosynthesis[59].胡黃連苦苷由一個單萜類起源的環(huán)烯醚萜骨架組成。根據(jù)功能部分，胡黃連苦苷分為胡黃連苦苷I和II。胡黃連苦苷I有肉桂酸部分，而胡黃連苦苷II有香子蘭酸鹽部分。肉桂酸和香子蘭酸鹽部分源于PP途徑(圖7)。肉桂酸的合成需要苯丙氨酸解氨酶(PAL)作用于在苯丙氨酸，而咖啡酰氧基-CoA3-O甲基轉(zhuǎn)移酶(COMT)是合成香子蘭酸鹽的關(guān)鍵酶。Iridoidmoietyisderivedfromgeranylpyrophosphate(GPP)(Figure7).GPPissynthesizedbysequentialheadtotailadditionofisopentenylpyrophosphate(IPP)anditsallelicisomerdimethylallylpyrophosphate(DMAPP).ThusMVA,MEPandPPareregardedascentralpathwaysforthesynthesisofpicrosides.MVApathwaystartsfromthecondensationofacetyl-CoA[67,68],whereasMEPpathwayneedspyruvateandglyceraldehyde3-phosphate[69,70].BiosynthesisofpicrosidesinvolvessynthesisofiridoidmoietyfromGPPthroughseriesofoxidationandcyclizationstepsfollowedbythecondensationofglucosemoietyandcinnamate/vanillatewithiridoidunit(Figure7).環(huán)烯醚萜苷部分來源于香葉基焦磷酸鹽(GPP)(圖7)。GPP是由連續(xù)的從頭到尾添加異戊烯基焦磷酸鹽(IPP)而合成，且它的等位基因異構(gòu)體是二甲基烯丙基焦磷酸酯(DMAPP)。胞質(zhì)MVA途徑和質(zhì)體局部MEP途徑合成IPP和DMAPP，通過這兩個通路交叉。因此MVA,MEP和PP被視為胡黃連苦苷合成的中央路徑方法。胡黃連苦苷的生物合成涉及來自GPP的環(huán)烯醚萜苷部分，在葡萄糖部分縮合、肉桂酸/香子蘭酸鹽與環(huán)烯醚萜苷單元之后經(jīng)過一系列氧化和環(huán)化步驟(圖7)。UsingBLASTanalysisagainsttheUniProtandKEGGdatabases,variousgenesassociatedwithMVA,MEPandPPpathwayswereidentified.RPKM-basedexpressionshowed2foldincreaseforseveralgenesofMEPpathwaysat15°Cascomparedtothoseat25°C(Figure8).Datawasinagreementwiththedataonpicrosidescontentthatshoweditsincreasedaccumulationat15°C(Figure3).WhileapreviousworkonP.kurrooaalsoshowedapositivecorrelationbetweenPkdxs(ageneofMEPpathway)andpicrosidesaccumulation,thepresentworkdetailedonallthegenesofMEPpathway.使用KEGG數(shù)據(jù)庫的BLAST分析，各種與MVA相關(guān)的基因和PP通路被鑒定。RPKM基礎(chǔ)的表達顯示MEP途徑一些基因在15℃條件下2倍增長（圖8）。15℃數(shù)據(jù)積累與胡黃連苦苷含量數(shù)據(jù)一致（圖3）。雖然以前對P.kurrooa的研究工作顯示Pkdxs(一個MEP通路基因)與胡黃連苦苷積累之間存在一種正相關(guān)，目前的工作是詳細描述MEP途徑的所有基因，強調(diào)它們對胡黃連苦苷積累的重要性（圖8）。RPKMdatashowedup-regulationofvariousgenesofMVAandPPpathwayat25°Cascomparedtothoseat15°C(Figure8,Additionalfile13).Theaccumulationofpicrosidesdecreasedat25°Cascomparedto15°C,whereasvariousgenesofPPpathwayexhibitedup-regulationat25°C.SincePPpathwayisimportantinsupplyingcinnamateandvallinateforpicrosidesbiosynthesis,anup-regulationofvariousgenesofthepathwayswereenvisionedat15°Cascomparedto25°C.RPKM數(shù)據(jù)顯示，與15℃相比，25℃下MVA和PP通路的各種基因上調(diào)（圖8，附件13）。與15℃相比，在25℃胡黃連苦苷的積累減少，而在25℃，PP通路各種基因顯示為上調(diào)。既然PP通路為胡黃連苦苷生物合成提供肉桂酸和香子蘭酸鹽是重要的，與25℃相比，預(yù)期通路的各種基因在15℃上調(diào)。Howevertheresultswereopposite,suggestingreroutingofthemetabolitestowardsthesynthesisofothermetabolitesat25°C.Andat15°C,theobservedexpressionofgenesoftheMVAandPPpathwaymightbesufficientenoughtomeettherequirementofcinnamateandvanillate.Infact,increasedactivityofPAL(ageneofPPpathway)inresponsetothermalstresswasconsideredasanacclamatoryresponseofcellstoheatstressinCitrulusvulgaris.然而，結(jié)果相反，說明在25℃代謝變更途徑成合成其他代謝物。15℃，MVA和PP途徑中觀測到的基因表達可能足夠充分滿足肉桂酸和香子蘭酸鹽的需要。事實上，

Citrulusvulgaris中，

PAL（PP途徑的一個基因）活性增加反映了熱壓力被認為是細胞對熱壓力的精彩回應(yīng)。轉(zhuǎn)錄組數(shù)據(jù)識別細胞色素P450(CYPs)和糖基轉(zhuǎn)移酶(GTs)作為迄今為止未知的胡黃連苦苷生物合成相關(guān)的基因TenCYPsshowedmorethantwofoldincreaseintheexpressionand8CYPsexhibiteddown-regulation,respectivelyat15°Cascomparedtoat25°C.Increasedpicrosidescontent(Figure3)andup-regulationofCYPsat15°CsuggestedthesetobethepossiblecandidatesassociatedwithpicrosidesbiosynthesisthroughtheirpossibleroleincyclizationofGPPandiridoidmoietyasindicatedinFigure7.在15℃與25℃相比,在表達中10CYPs顯示比2倍增加更多，8CYPs顯示下調(diào)。在15℃胡黃連苦苷含量增加（圖3）和CYPs上調(diào)說明這些可能通過在GPP與環(huán)烯醚萜部分環(huán)化作用過程中可能的作用與胡黃連苦苷生物合成相關(guān)（圖7）。實驗驗證RPKM數(shù)據(jù)通過反轉(zhuǎn)錄聚合酶連鎖反應(yīng)(RT-PCR)TheexpressionprofilesobtainedthroughRPKMvalueswereexperimentallyvalidatedthroughRT-PCRusing19genesbelongingtoMEP,MVAandPPpathways.RT-PCRdatashowedthatallthe7genesofMEPpathwayshowedup-regulationat15°CandthesimilartrendwasobtainedbyRPKMdataaswell(Figure8,Figure11)通過使用屬于MEP、MVA和PP途徑的19個基因進行RT-PCR實驗驗證從RPKM值獲得的表達譜數(shù)據(jù)。RT-PCR數(shù)據(jù)顯示MEP途徑的所有7個基因顯示上調(diào)在15℃，在RPKM數(shù)據(jù)中也有相似趨勢（圖8，圖11）。RT-PCRshowedup-regulationofHMGR,mevalonatekinase(MVK)andphosphomevalonatekinase(PMK)ofMVApathwayandCOMTofPPpathwayat15°C.However,RPKMdataexhibitedup-regulationofthesegenesat25°C.Expressionofacetyl-CoAacetyltransferase(ofMVApathway)didnot