劉涼琴, 宋愛(ài)萍, 張永俠, 原海燕, 黃蘇珍, 劉兆磊,①, 顧春筍,①
〔1. 南京農(nóng)業(yè)大學(xué)園藝學(xué)院, 江蘇 南京 210095; 2. 江蘇省中國(guó)科學(xué)院植物研究所(南京中山植物園), 江蘇 南京 210014〕
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馬藺根系響應(yīng)Cd脅迫的miRNA高通量測(cè)序分析
劉涼琴1, 宋愛(ài)萍1, 張永俠2, 原海燕2, 黃蘇珍2, 劉兆磊1,①, 顧春筍2,①
〔1. 南京農(nóng)業(yè)大學(xué)園藝學(xué)院, 江蘇 南京 210095; 2. 江蘇省中國(guó)科學(xué)院植物研究所(南京中山植物園), 江蘇 南京 210014〕
為了解Cd脅迫下馬藺〔Irislacteavar.chinensis(Fisch.) Koidz.〕根系miRNA的表達(dá)模式,采用高通量測(cè)序法對(duì)100 μmol·L-1Cd脅迫0(CK)和24 h(Cd)后馬藺根系的sRNA文庫(kù)(分別為CK和Cd文庫(kù))進(jìn)行分析,篩選出顯著差異表達(dá)的miRNA,并對(duì)這些miRNA的靶基因功能進(jìn)行預(yù)測(cè);在此基礎(chǔ)上,采用qRT-PCR技術(shù)對(duì)部分miRNA及其靶基因的表達(dá)模式進(jìn)行驗(yàn)證。結(jié)果表明:在CK和Cd文庫(kù)中,未注釋的sRNA序列較多,分別占各自sRNA特異序列總數(shù)的86.4%和80.5%;在已注釋的sRNA序列中,miRNA所占比例最低(分別為0.3%和0.5%),而rRNA所占比例最高(分別為9.4%和11.8%);2個(gè)文庫(kù)中的sRNA長(zhǎng)度主要為21~24 nt,且均以21 nt為最多。從Cd脅迫下馬藺根系sRNA中共篩選出32個(gè)顯著差異表達(dá)的miRNA,其中20個(gè)miRNA表達(dá)量下調(diào)(分別屬于miR165、miR166、miR167、miR168、miR390和miR396家族),12個(gè)miRNA表達(dá)量上調(diào)。功能預(yù)測(cè)結(jié)果表明:這些miRNA靶基因的功能主要集中在生物學(xué)過(guò)程、細(xì)胞組分和分子功能3個(gè)方面;而從KEGG通路富集分析看,富集在核糖體、氨基酸生物合成和碳代謝3個(gè)通路上的差異表達(dá)miRNA的靶基因數(shù)分別為122、88和82個(gè)。qRT-PCR驗(yàn)證結(jié)果表明:在CK和Cd文庫(kù)間,11個(gè)差異表達(dá)miRNA及8個(gè)靶基因的相對(duì)表達(dá)量均有顯著差異(P<0.05);其中,11個(gè)miRNA相對(duì)表達(dá)量的上調(diào)和下調(diào)趨勢(shì)與上述篩選結(jié)果一致,并且miRNA相對(duì)表達(dá)量上調(diào)時(shí),其靶基因的相對(duì)表達(dá)量下調(diào),反之亦然,說(shuō)明Cd脅迫條件下馬藺根系的miRNA負(fù)調(diào)控其靶基因的表達(dá),并且這些靶基因主要參與編碼轉(zhuǎn)錄因子、HD-ZIP蛋白和信號(hào)蛋白等過(guò)程。
馬藺; Cd脅迫; miRNA; 靶基因; 高通量測(cè)序; 差異表達(dá)
MicroRNA(miRNA)為一類單鏈、非編碼的小分子RNA,長(zhǎng)度為20~24 nt,在基因表達(dá)調(diào)控過(guò)程中起關(guān)鍵作用[1-2]。miRNA由含有莖環(huán)結(jié)構(gòu)的單鏈RNA前體通過(guò)DCL1加工形成[1];成熟的miRNA可與靶基因結(jié)合,實(shí)現(xiàn)對(duì)靶基因的抑制或切割[3];miRNA在植物發(fā)育過(guò)程中扮演重要角色,能夠調(diào)控根的萌發(fā)和生長(zhǎng)[4],并可控制葉片形態(tài)和極性[5]及花芽分化[6]等。擬南芥〔Arabidopsisthaliana(Linn.) Heynh.〕中的miR167通過(guò)調(diào)節(jié)靶基因ARF6和ARF8的表達(dá)控制體細(xì)胞胚的發(fā)生[7];硝酸鹽脅迫下,miR167通過(guò)顯著提高靶基因ARF8的表達(dá)量促進(jìn)側(cè)根的生長(zhǎng)發(fā)育[8];棉花(GossypiumhirsutumLinn.)中的miRNVL5通過(guò)介導(dǎo)GhCHR基因的表達(dá)提高植株的耐鹽性,同時(shí)GhCHR基因的表達(dá)量也顯著增加[9]。
隨著工業(yè)化進(jìn)程的加快,大量耕地受到重金屬污染。鎘(Cd)是毒性最強(qiáng)的重金屬元素之一,土壤中Cd含量超標(biāo)可使農(nóng)作物的可食部分中累積大量的Cd,嚴(yán)重影響農(nóng)作物的品質(zhì),并威脅人類健康[10]。miRNA能夠參與植物對(duì)重金屬脅迫的響應(yīng)過(guò)程,并在植物適應(yīng)重金屬脅迫方面發(fā)揮重要作用。Gielen等[11]發(fā)現(xiàn),擬南芥、歐洲油菜(BrassicanapusLinn.)、水稻(OryzasativaLinn.)和煙草(NicotianatabacumLinn.)等植物中的miRNA對(duì)Cd、As、Al和Hg等重金屬脅迫均非常敏感;Xie等[12]發(fā)現(xiàn),Cd脅迫下歐洲油菜的多個(gè)miRNA表達(dá)量上升;Zhang等[13]也發(fā)現(xiàn),Cd脅迫下,miR395超表達(dá)可阻止Cd進(jìn)入歐洲油菜的地上部分,提高其對(duì)Cd的耐性;水稻中有19個(gè)miRNA可響應(yīng)Cd脅迫,并且大多數(shù)miRNA能夠參與轉(zhuǎn)錄調(diào)控、信號(hào)轉(zhuǎn)導(dǎo)及脅迫響應(yīng)等過(guò)程[14-16];Zhou等[17]從Cd脅迫下的蒺藜苜蓿(MedicagotruncatulaGaertn.)中鑒定出38個(gè)未知miRNA,其中miR529、miR319、miR393和miR171表達(dá)量上調(diào),而miR398表達(dá)量則下調(diào);Sunkar等[18]認(rèn)為,CSD1和CSD2是擬南芥中miR398的靶基因,受miR398負(fù)調(diào)控表達(dá),CSD1和CSD2基因表達(dá)量的上調(diào)能夠減輕由Cd脅迫引起的氧化脅迫。上述研究結(jié)果均顯示:miRNA介導(dǎo)的基因表達(dá)在植物抵御重金屬脅迫過(guò)程中具有不可替代的作用。WAK基因是miR604的靶基因之一,參與響應(yīng)重金屬脅迫(Cu、Al、Zn)和植物防御機(jī)制[19-20];miR167和miR159的靶基因分別編碼NRAMP金屬離子轉(zhuǎn)運(yùn)蛋白和ABC轉(zhuǎn)運(yùn)蛋白,二者在重金屬離子吸收和平衡方面具有關(guān)鍵作用[21-23]。
馬藺〔Irislacteavar.chinensis(Fisch.) Koidz.〕為重金屬超富集植物[24],是研究植物對(duì)重金屬抗逆機(jī)制的重要物種之一。雖然目前已經(jīng)從馬藺中克隆獲得多個(gè)Cd耐性相關(guān)基因,并對(duì)其基因在Cd脅迫下的表達(dá)特性進(jìn)行了研究[25-26],但有關(guān)馬藺miRNA調(diào)控植株響應(yīng)和適應(yīng)重金屬脅迫的研究尚未見(jiàn)報(bào)道。
鑒于此,作者采用水培法、用100 μmol·L-1Cd培養(yǎng)馬藺,采集處理0和24 h的根系,分別提取總RNA并構(gòu)建sRNA文庫(kù);對(duì)sRNA文庫(kù)進(jìn)行高通量測(cè)序,對(duì)響應(yīng)Cd脅迫下差異表達(dá)的miRNAs進(jìn)行分析并預(yù)測(cè)其靶基因的功能;采用實(shí)時(shí)熒光定量PCR(qRT-PCR)技術(shù)對(duì)預(yù)測(cè)結(jié)果進(jìn)行驗(yàn)證,以期為馬藺根系miRNA應(yīng)答重金屬脅迫機(jī)制(尤其是應(yīng)答Cd脅迫機(jī)制)的深入研究奠定基礎(chǔ)。
1.1材料
使用的馬藺種子由江蘇省中國(guó)科學(xué)院植物研究所鳶尾課題組提供。選取飽滿的當(dāng)年生馬藺種子,用質(zhì)量分?jǐn)?shù)0.5%NaClO溶液消毒20 min,用自來(lái)水沖洗干凈并浸種催芽;種子萌發(fā)后,以石英砂(用自來(lái)水清洗干凈)為栽培基質(zhì)進(jìn)行播種;待幼苗株高約10 cm時(shí),選取長(zhǎng)勢(shì)一致的幼苗進(jìn)行脅迫實(shí)驗(yàn)[27]。
1.2方法
1.2.1Cd脅迫處理方法采用1/2Hoagland營(yíng)養(yǎng)液對(duì)幼苗進(jìn)行預(yù)培養(yǎng),幼苗用泡沫板固定并培養(yǎng)在塑料瓶中,每瓶3株幼苗,共6瓶18株。預(yù)培養(yǎng)1周后,向1/2Hoagland營(yíng)養(yǎng)液中加入CdCl2固體粉末,使?fàn)I養(yǎng)液中Cd終濃度達(dá)100 μmol·L-1;在培養(yǎng)0(CK)和24 h時(shí)分別采集其中3瓶幼苗的根系(每瓶視為1個(gè)重復(fù)),將同一塑料瓶中的3株幼苗根系混勻[28],立即用液氮速凍,并置于-80 ℃超低溫冰箱中保存、備用。
1.2.2總RNA的提取及sRNA文庫(kù)的構(gòu)建采用RNAisoTMPlus〔寶生物工程(大連)有限公司〕提取Cd脅迫0和24 h的馬藺根系總RNA并構(gòu)建sRNA文庫(kù),分別命名為CK文庫(kù)和Cd文庫(kù);采用Agilent 2100 Bioanalyzer和ABI StepOnePlusTMReal-Time PCR System對(duì)sRNA文庫(kù)進(jìn)行質(zhì)量檢測(cè),并用Illumina HiSeqTM2500超高通量測(cè)序系統(tǒng)進(jìn)行高通量測(cè)序,相關(guān)檢測(cè)由上海歐易生物醫(yī)學(xué)科技有限公司完成。
通過(guò)Base Callin將原始圖像數(shù)據(jù)轉(zhuǎn)換為序列數(shù)據(jù),即為原始數(shù)據(jù),并保存在“Fastq”文件中。為獲得高質(zhì)量的序列,從原始序列數(shù)據(jù)中去除接頭序列和質(zhì)量值Q≤5的堿基數(shù)占整個(gè)reads的50%以上的低質(zhì)量序列,將獲得的干凈序列(clean reads)用于數(shù)據(jù)分析。miRNA表達(dá)量采用TPM(transcripts per million)校準(zhǔn)[29],計(jì)算公式為:miRNA在文庫(kù)中的歸一化表達(dá)量=(Read count×106)/Total read count。若2個(gè)文庫(kù)中任何1個(gè)miRNA基因的表達(dá)量在歸一化后為0,則被修改為0.01;若2個(gè)文庫(kù)中任何1個(gè)miRNA基因的表達(dá)量在歸一化后小于1,則表示其表達(dá)量較低,不宜進(jìn)行差異表達(dá)分析。根據(jù)公式“差異倍數(shù)(fold change)=log2(Cd文庫(kù)中miRNA的歸一化表達(dá)量/CK文庫(kù)中miRNA的歸一化表達(dá)量)”計(jì)算CK與Cd文庫(kù)miRNA表達(dá)的差異倍數(shù)。
采用Allen等[31]的方法,根據(jù)同源性對(duì)差異表達(dá)的miRNA進(jìn)行靶基因預(yù)測(cè)[32]。將成熟的miRNA序列用于靶基因序列推測(cè),并對(duì)靶基因進(jìn)行GO功能分類和KEGG通路富集分析。
1.2.4差異表達(dá)miRNA及其靶基因表達(dá)模式的qRT-PCR驗(yàn)證在CK和Cd文庫(kù)中隨機(jī)選擇11個(gè)差異表達(dá)miRNA及7個(gè)差異表達(dá)miRNA的靶基因,采用qRT-PCR技術(shù)對(duì)其表達(dá)模式進(jìn)行驗(yàn)證。以成熟的miRNA序列為引物(表1),采用PrimeScript miRNA qPCR Starter Kit Ver.2.0〔寶生物工程(大連)有限公司〕對(duì)總RNA進(jìn)行反轉(zhuǎn)錄,以IlTIP41基因?yàn)閮?nèi)參基因[33];每個(gè)重復(fù)的總RNA樣品均進(jìn)行3次qRT-PCR擴(kuò)增(視為3個(gè)技術(shù)性重復(fù))。擴(kuò)增程序?yàn)椋?95 ℃預(yù)變性120 s; 95 ℃變性10 s、 55 ℃退火15 s、72 ℃延伸20 s,共40個(gè)循環(huán)。基因的相對(duì)表達(dá)量根據(jù)2-ΔΔCT計(jì)算[34],其中,CT值為每個(gè)反應(yīng)管內(nèi)熒光信號(hào)達(dá)到設(shè)定閾值時(shí)所需要的循環(huán)數(shù)。
表1用于馬藺根系部分miRNA及其靶基因qRT-PCR擴(kuò)增的引物序列
Table 1Primer sequences used for qRT-PCR amplification of some miRNA from root ofIrislacteavar.chinensis(Fisch.) Koidz. and their target genes
引物Primer序列(5'→3')Sequence(5'→3')miR165aTCGGACCAGGCTTCATCCCCCmiR166TCGGACCAGGCTTCATTCCCCmiR167d-5pTGAAGCTGCCAGCATGATCTGmiR168aTCGCTTGGTGCAGATCGGGACmiR396fTTCCACAGCTTTCTTGAACTGmiR159aTTTGGACTGAAGGGAGCTCTAmiR535TGACAACGAGAGAGAGCACGCmiR894CGTTTCACGTCGGGTTCACCmiR1511-3pACCTGGCTCTGATACCATAACmiR5139AAACCTGGCTCTGATACCAmiR8155TAACCTGGCTCTGATACCAmiR160gTGCCTGGCTCCCTGTATGCCAmiR166iTCGGACCAGGCTTCATTCCC
2.1Cd脅迫條件下馬藺根系sRNA的高通量測(cè)序結(jié)果分析
高通量測(cè)序結(jié)果表明:馬藺根系CK和Cd文庫(kù)中sRNA的原始序列(raw reads)分別有6 961 617和 7 459 170條;去除接頭序列、poly-A序列、≤18 nt序列和低質(zhì)量序列后,干凈序列分別有4 819 714和4 641 223條。在CK和Cd文庫(kù)中,sRNA的特異序列分別有1 326 618和1 146 173條(表2),其中,能與基因組比對(duì)成功的sRNA特異序列分別有519 023和532 832條,分別占各自sRNA特異序列總數(shù)的39.1%和46.5%。
由表2可見(jiàn):CK和Cd文庫(kù)中sRNA類型較多,包括miRNA、rRNA、snRNA、snoRNA、tRNA和重復(fù)序列。其中,CK和Cd文庫(kù)中未注釋的sRNA序列分別有1 145 700和922 152條,分別占各自sRNA特異序列總數(shù)的86.4%和80.5%;已注釋的sRNA序列分別有180 918和224 021條。在已注釋的sRNA中,rRNA最多,分別占CK和Cd文庫(kù)中sRNA特異序列總數(shù)的9.4%和11.8%;其次為tRNA,分別占各自sRNA特異序列總數(shù)的2.2%和3.1%;miRNA最少,分別僅占各自sRNA特異序列總數(shù)的0.3%和0.5%。
對(duì)馬藺根系CK和Cd文庫(kù)中sRNA長(zhǎng)度的分析結(jié)果見(jiàn)圖1。由圖1可以看出:在CK和Cd文庫(kù)中,長(zhǎng)度為21 nt的sRNA所占比例最高,分別為29.1%和34.0%; 其次是長(zhǎng)度為24 nt的sRNA。 總體上看,在馬藺根系的CK和Cd文庫(kù)中,長(zhǎng)度為21~24 nt的sRNA所占比例較高,分別為75.7%和80.9%。
表2Cd脅迫條件下馬藺根系sRNA高通量測(cè)序結(jié)果的分析1)
Table 2Analysis on result of high throughput sequencing of sRNA in root ofIrislacteavar.chinensis(Fisch.) Koidz. under Cd stress1)
sRNA類型TypeofsRNACK文庫(kù) CKlibrary數(shù)量Number比例/%PercentageCd文庫(kù) Cdlibrary數(shù)量Number比例/%PercentagemiRNA 32570.3 53120.5rRNA1241079.413581911.8snRNA55280.4106640.9snoRNA72090.5137041.2tRNA296912.2357633.1重復(fù)序列Repeatsequence111260.8227592.0未注釋序列Unannotationsequence114570086.492215280.5總計(jì)Total1326618100.01146173100.0
1)CK: 100 μmol·L-1Cd脅迫0 h Stressed by 100 μmol·L-1Cd for 0 h; Cd: 100 μmol·L-1Cd脅迫24 h Stressed by 100 μmol·L-1Cd for 24 h.
□: CK文庫(kù)(100 μmol·L-1Cd脅迫0 h) CK library (stressed by 100 μmol·L-1 Cd for 0 h); ■: Cd文庫(kù)(100 μmol·L-1Cd脅迫24 h) Cd library (stressed by 100 μmol·L-1 Cd for 24 h).圖1 Cd脅迫下馬藺根系CK和Cd文庫(kù)中不同長(zhǎng)度sRNA的比例Fig. 1 Analysis on percentage of sRNA with different lengths in CK and Cd libraries from root ofIris lactea var. chinensis (Fisch.) Koidz. under Cd stress
2.2Cd脅迫條件下馬藺根系中顯著差異表達(dá)miRNA的篩選結(jié)果
2.3Cd脅迫條件下馬藺根系中差異表達(dá)miRNA靶基因功能的預(yù)測(cè)結(jié)果
利用GO功能分類對(duì)Cd脅迫條件下馬藺根系中差異表達(dá)的miRNA靶基因進(jìn)行功能預(yù)測(cè),結(jié)果(圖2)表明:大部分差異表達(dá)miRNA靶基因的功能主要集中在生物學(xué)過(guò)程、細(xì)胞組分和分子功能3個(gè)方面。
在生物學(xué)過(guò)程中,差異表達(dá)miRNA靶基因的功能主要集中在氧化還原過(guò)程(oxidation-reduction process)、 以DNA為模板的轉(zhuǎn)錄調(diào)控 (regulation of transcription,DNA templated)和蛋白質(zhì)磷酸化(protein phosphorylation)等過(guò)程(圖2-A);在細(xì)胞組分中,細(xì)胞核(nucleus)、質(zhì)膜(plasma membrane)和線粒體(mitochondrion)中的差異表達(dá)miRNA靶基因較多(圖2-B);而在分子功能中,多數(shù)差異表達(dá)miRNA靶基因與結(jié)合功能有關(guān),與ATP結(jié)合有關(guān)的miRNA靶基因最多,其次是與鋅離子結(jié)合的靶基因(圖2-C)。
表3Cd脅迫條件下馬藺根系中顯著差異表達(dá)miRNA的表達(dá)特性分析
Table 3Analysis on expression character of miRNA with significantly differential expression in root ofIrislacteavar.chinensis(Fisch.) Koidz. under Cd stress
miRNA名稱1)miRNAname1)在不同文庫(kù)中的表達(dá)量2) Expressionindifferentlibraries2)CK文庫(kù)CKlibraryCd文庫(kù)Cdlibrary差異倍數(shù)Foldchange 調(diào)控類型 TypeofregulationmiR165a 787.39 175.60-2.16 下調(diào)Down-regulatedmiR166j6.221.51-2.05 下調(diào)Down-regulatedmiR166i487.37124.32-1.97 下調(diào)Down-regulatedmiR166a38190.449757.13-1.97 下調(diào)Down-regulatedmiR16637979.019750.23-1.96 下調(diào)Down-regulatedmiR166e-3p135.2835.55-1.93 下調(diào)Down-regulatedmiR166j-3p85.0722.62-1.91 下調(diào)Down-regulatedmiR166u259.1471.32-1.86 下調(diào)Down-regulatedmiR166m97.7229.09-1.75 下調(diào)Down-regulatedmiR166b118.0637.06-1.67 下調(diào)Down-regulatedmiR165a-3p3.321.08-1.62 下調(diào)Down-regulatedmiR166g-3p143.5847.19-1.61 下調(diào)Down-regulatedmiR166h-3p81853.6127682.79-1.56 下調(diào)Down-regulatedmiR166n44.6115.30-1.54 下調(diào)Down-regulatedmiR167d-5p6.222.37-1.39 下調(diào)Down-regulatedmiR166k41.0817.02-1.27 下調(diào)Down-regulatedmiR390a4.151.72-1.27 下調(diào)Down-regulatedmiR396f1175.80515.17-1.19 下調(diào)Down-regulatedmiR168a-3p117.8553.43-1.14 下調(diào)Down-regulatedmiR168a143.3769.59-1.04 下調(diào)Down-regulatedmiR53527.5958.611.09 上調(diào)Up-regulatedmiR535a28.6361.411.10 上調(diào)Up-regulatedmiR159a736.971605.401.12 上調(diào)Up-regulatedmiR81555.8112.711.13 上調(diào)Up-regulatedmiR51398.0918.101.16 上調(diào)Up-regulatedmiR1511-3p3.327.761.22 上調(diào)Up-regulatedmiR159-3p2.495.821.22 上調(diào)Up-regulatedmiR159b31.3374.121.24 上調(diào)Up-regulatedmiR160g1.243.881.64 上調(diào)Up-regulatedmiR89420.5478.211.93 上調(diào)Up-regulatedmiR396b-3p3.7314.441.95 上調(diào)Up-regulatedmiR29160.412.372.51 上調(diào)Up-regulated
2)為歸一化后的表達(dá)量 Expression after normalization. CK: 100 μmol·L-1Cd脅迫0 h Stressed by 100 μmol·L-1Cd for 0 h; Cd: 100 μmol·L-1Cd脅迫24 h Stressed by 100 μmol·L-1Cd for 24 h.
圖2 Cd脅迫條件下馬藺根系中涉及生物學(xué)過(guò)程(A)、細(xì)胞組分(B)和分子功能(C)的差異表達(dá)miRNA靶基因的GO功能分類結(jié)果Fig. 2 Result of GO function classification on target genes of differential expression miRNA related to biological process (A), cellular component (B) and molecular function (C) in root of Iris lactea var. chinensis (Fisch.) Koidz. under Cd stress
對(duì)馬藺根系中差異表達(dá)的miRNA靶基因進(jìn)行KEGG通路富集分析,結(jié)果見(jiàn)圖3。結(jié)果顯示:富集差異表達(dá)miRNA靶基因數(shù)量最多的通路依次為核糖體(ribosome)、氨基酸生物合成(biosynthesis of amino acids)和碳代謝(carbon metabolism),3個(gè)通路富集的差異表達(dá)miRNA靶基因數(shù)分別為122、88和82個(gè)。
圖3 Cd脅迫條件下馬藺根系中差異表達(dá)miRNA靶基因的KEGG通路富集分析的結(jié)果Fig. 3 Result on KEGG pathway enrichment analysis of target genes of differential expression miRNA in root ofIris lactea var. chinensis (Fisch.) Koidz. under Cd stress
2.4Cd脅迫條件下馬藺根系中差異表達(dá)miRNA及其靶基因表達(dá)模式的qRT-PCR驗(yàn)證結(jié)果
2.4.1差異表達(dá)miRNA表達(dá)模式的驗(yàn)證結(jié)果對(duì)Cd脅迫條件下馬藺根系CK和Cd文庫(kù)中5個(gè)下調(diào)表達(dá)的miRNA(包括miR165a、miR166、miR167d-5p、miR168a和miR396f)和6個(gè)上調(diào)表達(dá)的miRNA(包括miR159a、miR535、miR894、miR1511-3p、miR5139和miR8155)進(jìn)行qRT-PCR驗(yàn)證,結(jié)果見(jiàn)圖4。結(jié)果顯示:CK和Cd文庫(kù)間供試11個(gè)差異表達(dá)miRNA的相對(duì)表達(dá)量均有顯著差異(P<0.05);雖然這11個(gè)miRNA的表達(dá)水平差異與表3中的數(shù)據(jù)并不完全匹配,但是表達(dá)的上調(diào)和下調(diào)趨勢(shì)一致,說(shuō)明高通量測(cè)序結(jié)果可靠。
2.4.2差異表達(dá)miRNA靶基因表達(dá)模式的驗(yàn)證結(jié)果對(duì)Cd脅迫下馬藺根系CK和Cd文庫(kù)中7個(gè)差異表達(dá)miRNA(包括miR396f、miR165a、miR166、miR166i、 miR160g、 miR1511-3p和miR159a)的8個(gè)靶基因(其中miR159a有2個(gè)靶基因)的表達(dá)模式進(jìn)行qRT-PCR驗(yàn)證,結(jié)果見(jiàn)圖5。結(jié)果表明:miR396f的靶基因comp281813編碼生長(zhǎng)調(diào)節(jié)因子5,miR165a的靶基因comp250130編碼HD-ZIP蛋白ATHB-15,miR166的靶基因comp329160編碼HD-ZIP蛋白HOX32,miR166i的靶基因comp311396編碼轉(zhuǎn)錄因子PCF6,miR160g的靶基因comp357989編碼生長(zhǎng)素響應(yīng)因子17,miR1511-3p的靶基因comp120421編碼細(xì)胞分裂蛋白SepF,miR159a的靶基因comp239971和comp350997分別編碼細(xì)胞色素C氧化酶亞基3和轉(zhuǎn)錄因子GAMYB。
由圖5還可見(jiàn):在CK和Cd文庫(kù)間,靶基因comp311396的相對(duì)表達(dá)量無(wú)顯著差異,其余靶基因的相對(duì)表達(dá)量均存在顯著差異。另外,在CK文庫(kù)中未檢測(cè)到靶基因comp281813和comp250130的表達(dá),而且在Cd文庫(kù)中也未檢測(cè)到靶基因comp120421和comp239971的表達(dá)。
□: CK文庫(kù)(100 μmol·L-1Cd脅迫0 h) CK library (stressed by 100 μmol·L-1Cd for 0 h);
■: Cd文庫(kù)(100 μmol·L-1Cd脅迫24 h) Cd library (stressed by 100 μmol·L-1Cd for 24 h).
不同小寫(xiě)字母表示不同文庫(kù)間同一類miRNA的相對(duì)表達(dá)量有顯著差異(P<0.05) Different small letters indicate the significant difference in relative expressions of the same miRNA among different libraries.
圖4Cd脅迫條件下馬藺根系sRNA文庫(kù)中11個(gè)差異表達(dá)miRNA的相對(duì)表達(dá)量比較
Fig. 4Comparison on relative expression of eleven differential expression miRNAs in sRNA libraries from root ofIrislacteavar.chinensis(Fisch.) Koidz. under Cd stress
□: CK文庫(kù)(100 μmol·L-1Cd脅迫0 h) CK library (stressed by 100 μmol·L-1 Cd for 0 h); ■: Cd文庫(kù)(100 μmol·L-1Cd脅迫24 h) Cd library (stressed by 100 μmol·L-1 Cd for 24 h).
comp281813: miR396f的靶基因 Target gene of miR396f;comp250130: miR165a的靶基因 Target gene of miR165a;comp329160: miR166的靶基因 Target gene of miR166;comp311396: miR166i的靶基因 Target gene of miR166i;comp357989: miR160g的靶基因 Target gene of miR160g;comp120421: miR1511-3p的靶基因 Target gene of miR1511-3p;comp239971,comp350997: miR159a的靶基因 Target gene of miR159a. +: 未檢出 Undetected; *: 表示不同文庫(kù)間同一靶基因的相對(duì)表達(dá)量有顯著差異(P<0.05) Indicating the significant difference in relative expressions of the same target gene among different libraries.
圖5Cd脅迫條件下馬藺根系sRNA文庫(kù)中7個(gè)差異表達(dá)miRNA的靶基因相對(duì)表達(dá)量的比較
Fig. 5Comparison on relative expression of target genes of seven differential expression miRNAs in sRNA libraries from root ofIrislacteavar.chinensis(Fisch.) Koidz. under Cd stress
不同植物中的sRNA存在一定差異。本研究中,100 μmol·L-1Cd脅迫0(對(duì)照)和24 h,馬藺根系的sRNA長(zhǎng)度均以21和24 nt 為主,并且前者最多。相關(guān)研究結(jié)果顯示:蘿卜(RaphanussativusLinn.)中的sRNA長(zhǎng)度以21和24 nt為主[35];番茄(SolanumlycopersicumLinn.)[36]和枳〔Poncirustrifoliata(Linn.) Raf.〕[37]的sRNA長(zhǎng)度以21 nt為最多,而擬南芥[38]中的sRNA長(zhǎng)度則以24 nt為最多。表明不同植物的sRNA長(zhǎng)度和含量存在差異,可能與植物種類及參與sRNA生物合成的酶不同有關(guān)。高通量測(cè)序結(jié)果顯示:馬藺根系CK和Cd文庫(kù)中sRNA類型豐富;2個(gè)文庫(kù)的已注釋sRNA中,rRNA比例均最高(9.4%和11.8%),miRNA比例均最低(0.3%和0.5%),而未注釋的sRNA比例均在80%以上,說(shuō)明馬藺根系中未知的sRNA數(shù)量較多,有待進(jìn)一步深入研究。
研究結(jié)果表明:100 μmol·L-1Cd脅迫條件下,馬藺根系中共有32個(gè)miRNA顯著差異表達(dá),其中miR165a、miR166、miR167d-5p、miR168a和miR396f等20個(gè)miRNA下調(diào)表達(dá),其余12個(gè)miRNA上調(diào)表達(dá)。在Cd脅迫條件下,水稻的miR166、miR168和miR396下調(diào)表達(dá)[14],蒺藜苜蓿的miR166也下調(diào)表達(dá)[17];在非生物脅迫(鹽、干旱和冷害脅迫)條件下,擬南芥的miR168和miR396上調(diào)表達(dá)[39]。Cd脅迫條件下,不同植物同一家族miRNA的表達(dá)水平也存在差異[12,40-41]。馬藺根系中的miR159a和miR894受Cd脅迫誘導(dǎo)表達(dá),這與Zhang等[42]的研究結(jié)果一致。根據(jù)本研究結(jié)果及上述文獻(xiàn)報(bào)道,推測(cè)miR166、miR168和miR396可能介導(dǎo)了植物對(duì)各種非生物脅迫的響應(yīng),并且,植物體內(nèi)不同miRNA對(duì)非生物脅迫的響應(yīng)具有特異性,但許多miRNA的調(diào)控機(jī)制尚未明確,有待深入研究。
對(duì)Cd脅迫條件下馬藺根系中差異表達(dá)的miRNA靶基因功能的預(yù)測(cè)結(jié)果表明:7個(gè)差異表達(dá)miRNA的靶基因主要參與編碼轉(zhuǎn)錄因子、HD-ZIP蛋白和信號(hào)蛋白等。而對(duì)miRNA及其靶基因表達(dá)狀況的比較結(jié)果表明:當(dāng)miRNA表達(dá)水平上調(diào)時(shí),其靶基因的相對(duì)表達(dá)量降低;而當(dāng)miRNA表達(dá)水平下調(diào)時(shí),其靶基因的相對(duì)表達(dá)量則升高。說(shuō)明Cd脅迫條件下馬藺根系中這些差異表達(dá)的miRNA對(duì)其靶基因具有負(fù)調(diào)控作用,并說(shuō)明本研究對(duì)差異表達(dá)miRNA靶基因的預(yù)測(cè)結(jié)果可靠。HD-ZIP蛋白在植物器官發(fā)育過(guò)程中起重要作用[43-45],馬藺根系中miR166的靶基因編碼HD-ZIP HOX32蛋白,據(jù)此推測(cè)miR166可能參與馬藺的器官發(fā)育;馬藺根系中miR165a的靶基因編碼HD-ZIP ATHB-15蛋白,而該蛋白已被證實(shí)與植物響應(yīng)脅迫有關(guān)[46-47]。生長(zhǎng)素響應(yīng)因子ARF17在生長(zhǎng)素信號(hào)通路中發(fā)揮作用[18],該轉(zhuǎn)錄因子由馬藺miR160g的靶基因所編碼,能夠響應(yīng)Cd脅迫;而miR160家族不同成員通過(guò)調(diào)控ARFs響應(yīng)不同類型的環(huán)境脅迫[35,37,48],因而,miR160在調(diào)控植物生長(zhǎng)發(fā)育和植物響應(yīng)各種非生物脅迫過(guò)程中發(fā)揮重要作用,但不同植物的miR160對(duì)脅迫的響應(yīng)機(jī)制尚待進(jìn)一步探究。Cd脅迫條件下,馬藺根系中miR159a負(fù)調(diào)控2個(gè)靶基因(編碼轉(zhuǎn)錄因子GAMYB和細(xì)胞色素C氧化酶亞基3)的表達(dá),小麥(TriticumaestivumLinn.)根和葉中的miR159負(fù)調(diào)控靶基因MYB3的表達(dá)[49],擬南芥中的miR159抑制靶基因MYB33和MYB65的表達(dá)[50];此外,蘿卜根系中的miR159靶向調(diào)控ABC轉(zhuǎn)運(yùn)蛋白,而該轉(zhuǎn)運(yùn)蛋白在植物對(duì)重金屬離子的吸收與轉(zhuǎn)運(yùn)過(guò)程中發(fā)揮關(guān)鍵作用[51],據(jù)此推測(cè)miR159可能參與植物的應(yīng)激反應(yīng)。
綜合分析結(jié)果表明:馬藺根系中未注釋的sRNA序列較多,且在已注釋的sRNA序列中rRNA所占比例最高,而miRNA所占比例最低;其sRNA長(zhǎng)度主要集中在21~24 nt,并以21 nt為最多。通過(guò)高通量測(cè)序篩選出32個(gè)顯著差異表達(dá)的miRNA,其中,20個(gè)miRNA下調(diào)表達(dá),12個(gè)miRNA上調(diào)表達(dá)。馬藺根系miRNA靶基因的功能主要集中在生物學(xué)過(guò)程、細(xì)胞組分和分子功能3個(gè)方面;其miRNA均對(duì)靶基因的表達(dá)具有負(fù)調(diào)控作用,并且這些靶基因主要參與編碼轉(zhuǎn)錄因子、HD-ZIP蛋白和信號(hào)蛋白等過(guò)程。
[1]BARTEL D. MicroRNAs: genomics, biogenesis, mechanism and function[J]. Cell, 2004, 116: 281-297.
[2]VOINNET O. Origin, biogenesis, and activity of plant microRNAs[J]. Cell, 2009, 136: 669-687.
[3]RHOADES M W, REINHART B J, LIM L P, et al. Prediction of plant microRNA targets[J]. Cell, 2002, 110: 513-520.
[4]MARIN E, JOUANNET V, HERZ A, et al. miR390,ArabidopsisTAS3 tasiRNAs, and theirAUXINRESPONSEFACTORtargets define an autoregulatory network quantitatively regulating lateral root growth[J]. The Plant Cell, 2010, 22: 1104-1117.
[5]MALLORY A C, REINHART B J, JONES-RHOADES M W, et al. MicroRNA control ofPHABULOSAin leaf development: importance of pairing to the microRNA 5′ region[J]. The EMBO Journal, 2004, 23: 3356-3364.
[6]CHUCK G, MEELEY R, HAKE S. Floral meristem initiation and meristem cell fate are regulated by the maizeAP2 genesids1 andsid1 [J]. Development, 2008, 135: 3013-3019.
[7]SU Y H, LIU Y B, ZHOU C, et al. The microRNA167 controls somatic embryogenesis inArabidopsisthrough regulating its target genesARF6 andARF8[J]. Plant Cell, Tissue and Organ Culture, 2016, 124: 405-417.
[8]GIFFORD M L, DEAN A, GUTIERREZ R A, et al. Cell-specific nitrogen responses mediate developmental plasticity[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105: 803-808.
[9]GAO S, YANG L, ZENG H Q, et al. A cotton miRNA is involved in regulation of plant response to salt stress[J]. Scientific Reports, 2016, 6: e19736.
[10]FOWLER B A. Monitoring of human populations for early markers of cadmium toxicity: a review[J]. Toxicology and Applied Pharmacology, 2009, 238: 294-300.
[11]GIELEN H, REMANS T, VANGRONSVELD J, et al. MicroRNAs in metal stress: specific roles or secondary responses?[J]. International Journal of Molecular Sciences, 2012, 13: 15826-15847.
[12]XIE F L, HUANG S Q, GUO K, et al. Computational identifi-cation of novel microRNAs and targets inBrassicanapus[J]. FEBS Letters, 2007, 581: 1464-1474.
[13]ZHANG L W, SONG J B, SHU X X, et al. miR395 is involved in detoxification of cadmium inBrassicanapus[J]. Journal of Hazardous Materials, 2013, 250/251: 204-211.
[14]DINGYF,CHENZ,ZHUC.Microarray-basedanalysisof cadmium-responsive microRNAs in rice (Oryzasativa)[J]. Journal of Experimental Botany, 2011, 62: 3563-3573.
[15]MENGYJ,CHENDJ,MAXX,etal.Mechanismsof microRNA-mediated auxin signaling inferred from the rice mutantosaxr[J]. Plant Signaling and Behavior, 2010, 5: 252-254.
[16]LI T, LI H, ZHANG Y X, et al. Identification and analysis of seven H2O2-responsive miRNAs and 32 new miRNAs in the seedlings of rice (OryzasativaL. ssp.indica)[J]. Nucleic Acids Research, 2011, 39: 2821-2833.
[17]ZHOU Z S, HUANG S Q, YANG Z M. Bioinformatic identification and expression analysis of new microRNAs fromMedicagotruncatula[J]. Biochemical and Biophysical Research Communications, 2008, 374: 538-542.
[18]SUNKAR R, KAPOOR A, ZHU J K. Posttranscriptional induction of two Cu/Zn superoxide dismutase genes inArabidopsisis mediated by downregulation of miR398 and important for oxidative stress tolerance[J]. The Plant Cell, 2006, 18: 2051-2065.
[19]SIVAGURU M, EZAKI B, HE Z H, et al. Aluminum-induced gene expression and protein localization of a cell wall-associated receptor kinase in Arabidopsis[J]. Plant Physioligy, 2003, 132: 2256-2266.
[20]HOU X W, TONG H Y, SELBY J, et al. Involvement of a cell wall-associated kinase, WAKL4, in Arabidopsis mineral responses[J]. Plant Physiology, 2005, 139: 1704-1716.
[21]BOVET L, EGGMANN T, MEYLAND-BETTEX M, et al. Tran-script levels ofAtMRPsafter cadmium treatment: induction ofAtMRP3[J]. Plant, Cell and Environment, 2003, 26: 371-381.
[22]TALKEIN,HANIKENNEM,KRMERU. Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulatorArabidopsishalleri[J]. Plant Physiology, 2006, 142: 148-167.
[24]HANYL,HUANGSZ,GUJG,etal.Tolerance and accumulation of lead by species ofIrisL.[J]. Ecotoxicology, 2008, 17: 853-859.
[25]GU C S, LIU L Q, ZHAO Y H, et al. Overexpression ofIrislacteavar.chinensismetallothioneinllMT2aenhances cadmium tolerance inArabidopsisthaliana[J]. Ecotoxicology and Environmental Safety, 2014, 105: 22-28.
[26]GU C S, LIU L Q, DENG Y M, et al. The heterologous expression of theIrislacteavar.chinensistype 2 metallothioneinllMT2bgene enhances copper tolerance inArabidopsisthaliana[J]. Bulletin of Environmental Contamination and Toxicology, 2015, 94: 247-253.
[27]郭智, 黃蘇珍, 原海燕. Cd脅迫對(duì)馬藺和鳶尾幼苗生長(zhǎng)、Cd積累及微量元素吸收的影響[J]. 生態(tài)環(huán)境, 2008, 17(2): 651-656.
[28]TIAN S Q, GU C S, LIU L Q, et al. Transcriptome profiling ofLouisianairisroot and identification of genes involved in lead-stress response[J]. International Journal of Molecular Sciences, 2015, 16: 28087-28097.
[29]ZHOUL,CHEN J H,LI Z Z,et al.Integrated profiling of microRNAs and mRNAs: microRNAs located on Xq27.3 associate with clear cell renal cell carcinoma[J]. PLoS One, 2010, 5: e15224.
[30]KOZOMARA A, GRIFFITHS-JONES S. miRBase: annotating high confidence microRNAs using deep sequencing data[J]. Nucleic Acids Research, 2014, 42: 68-73.
[31]ALLENE,XIEZX, GUSTAFSONAM, et al. microRNA-directed phasing duringtrans-acting siRNA biogenesis in plants[J]. Cell, 2005, 121: 207-221.
[32]BONNET E, WUYTS J, ROUZé P, et al. Detection of 91 potential conserved plant microRNAs inArabidopsisthalianaandOryzasativaidentifies important target genes[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101: 11511-11516.
[33]GU C S, LIU L Q, DENG Y M, et al. Validation of reference genes for RT-qPCR normalization inIrislacteavar.chinensisleaves under different experimental conditions[J]. Scientia Horticulturae, 2014, 175: 144-149.
[34]汪仁, 蔡黎麗, 徐晟, 等. 石蒜Mg2+轉(zhuǎn)運(yùn)體基因LrMGT的克隆與分析[J]. 植物資源與環(huán)境學(xué)報(bào), 2014, 23(4): 1-7.
[35]SUN X C, XU L, WANG Y, et al. Identification of novel and salt-responsive miRNAs to explore miRNA-mediated regulatory network of salt stress response in radish (RaphanussativusL.)[J]. BMC Genomics, 2015, 16: 197.
[36]MOXON S, JING R C, SZITTYA G, et al. Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening[J]. Genome Research, 2008, 18: 1602-1609.
[37]ZHANG X N, LI X, LIU J H. Identification of conserved and novel cold-Responsive microRNAs in trifoliate orange (Poncirustrifoliata(L.) Raf.) using high-throughput sequencing[J]. Plant Molecular Biology Reporter, 2014, 32: 328-341.
[38]KASSCHAUKD, FAHLGRENN, CHAPMANEJ,etal.Genome-wide profiling and analysis ofArabidopsissiRNAs[J]. PLoS Biology, 2007, 5: e57.
[39]LIU H H, TIAN X, LI Y J, et al. Microarray-based analysis of stress-regulated microRNAs inArabidopsisthaliana[J]. RNA, 2008, 14: 836-843.
[40]XU L, WANG Y, ZHAI L L, et al. Genome-wide identification and characterization of cadmium-responsive microRNAs and their target genes in radish (RaphanussativusL.) roots[J]. Journal of Experimental Botany, 2013, 64: 4271-4287.
[41]FANG X L, ZHAO Y Y, MA Q B, et al. Identification and comparative analysis of cadmium tolerance-associated miRNAs and their targets in two soybean genotypes[J]. PLoS One, 2013, 8: e81471.
[42]ZHANG Q, ZHAO C Z, LI M, et al. Genome-wide identification ofThellungiellasalsugineamicroRNAs with putative roles in the salt stress response[J]. BMC Plant Biology, 2013, 13: 180.
[43]HAWKER N P, BOWMAN J L. Roles for class ⅢHD-ZipandKANADIgenes in Arabidopsis root development[J]. Plant Physiology, 2004, 135: 2261-2270.
[44]PRIGGEMJ,OTSUGAD,ALONSOJM, et al. Class Ⅲ homeodomain-leucine zipper gene family members have overlapping, antagonistic, and distinct roles in Arabidopsis development[J]. The Plant Cell, 2005, 17: 61-76.
[45]BARVKAR V T, PARDESHI V C, KALE S M, et al. Genome-wide identification and characterization of microRNA genes and their
targets in flax (Linumusitatissimum)[J]. Planta, 2013, 237: 1149-1161.
[46]AGALOU A, PURWANTOMO S, ?VERNS E, et al. A genome-wide survey ofHD-Zipgenes in rice and analysis of drought-responsive family members[J]. Plant Molecular Biology, 2008, 66: 87-103.
[47]DAI M Q, HU Y F, MA Q, et al. Functional analysis of riceHOMEOBOX4 (Oshox4) gene reveals a negative function in gibberellinresponses[J]. PlantMolecularBiology, 2008, 66: 289-301.
[48]ELDEM V, AK?AY U ?, OZHUNER E, et al. Genome-wide identification of miRNAs responsive to drought in peach (Prunuspersica) by high-throughput deep sequencing[J]. PLoS One, 2012, 7: e50298.
[49]QIU Z B, HAI B Z, GUO J L, et al. Characterization of wheat miRNAs and their target genes responsive to cadmium stress[J]. Plant Physiology and Biochemistry, 2016, 101: 60-67.
[50]ALLEN R S, LI J Y, STAHLE M I, et al. Genetic analysis reveals functional redundancy and the major target genes of theArabidopsismiR159 family[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104: 16371-16376.
[51]CAO F B, CHEN F, SUN H Y, et al. Genome-wide transcriptome and functional analysis of two contrasting genotypes reveals key genes for cadmium tolerance in barley[J]. BMC Genomics, 2014, 15: 611.
(責(zé)任編輯: 佟金鳳)
High throughput sequencing analysis on miRNA in root ofIrislacteavar.chinensisresponse to Cd stress
LIU Liangqin1, SONG Aiping1, ZHANG Yongxia2, YUAN Haiyan2, HUANG Suzhen2, LIU Zhaolei1,①, GU Chunsun2,①
(1. College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China; 2. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China),J.PlantResour. &Environ., 2016, 25(3): 1-11
In order to understand the expression pattern of miRNA in root ofIrislacteavar.chinensis(Fisch.) Koidz. under Cd stress, sRNA libraries (which is CK and Cd libraries, respectively) from root ofI.lacteavar.chinensisafter stressed by 100 μmol·L-1Cd for 0 (CK) and 24 h (Cd) were analyzed by method of high throughput sequencing analysis. Also, miRNA with significantly differential expression was screened, and functions of target genes of these miRNA were predicted. On this basis, expression pattern of some miRNA and their target genes were verified by qRT-PCR technology. The results show that in CK and Cd libraries, there are more unannotation sRNA sequences, accounting for 86.4% and 80.5% of total number of their own sRNA unique reads, respectively. Among annotation sRNA sequences, percentage of miRNA is the lowest with a value of 0.3% and 0.5%, respectively, while that of rRNA is the highest with a value of 9.4% and 11.8%, respectively. Length of sRNA in the two libraries is mainly 21-24 nt, and the most of them is 21 nt. Thirty-two miRNA with significantly differential expression are screened from sRNA in root ofI.lacteavar.chinensisunder Cd stress, in which, expression of twenty miRNA is down-regulated (belonging to miR165, miR166, miR167, miR168, miR390 and miR396 families, respectively), that of twelve miRNA is up-regulated. The result of function prediction indicates that functions of target genes of these miRNA are mainly concentrated in three aspects, i.e. biological process, cellular component and molecular function, while from KEGG pathway enrichment analysis, number of target gene of differential expression miRNA enriching in three pathways of ribosome, biosynthesis of amino acids and carbon metabolism is 122, 88 and 82, respectively. Verification result of qRT-PCR shows that between CK and Cd libaries, there are significantly differences in relative expression of eleven miRNA with differential expression and eight target genes (P<0.05). In which, up-regulated and down-regulated trends of relative expression of eleven miRNA are consistent with above screening result, and when relative expression of miRNA is up-regulated, that of its target gene is down-regulated, andviceversa, meaning that under Cd stress condition, miRNA from root ofI.lacteavar.chinensisnegatively regulates its target gene expression, and these target genes mainly are involved in processes of coding transcription factor, HD-ZIP protein and signaling protein, etc.
Irislacteavar.chinensis(Fisch.) Koidz.; Cd stress; miRNA; target gene; high throughput sequencing; differential expression
2016-06-07
國(guó)家自然科學(xué)基金資助項(xiàng)目(31301807; 31300436); 江蘇省自然科學(xué)基金資助項(xiàng)目(BK20130734)
劉涼琴(1990—),女,江蘇常州人,碩士研究生,主要從事觀賞植物分子育種研究。
E-mail: lzl@njau.edu.cn; chunsungu@126.com
Q946-33; S682.1+9
A
1674-7895(2016)03-0001-11
10.3969/j.issn.1674-7895.2016.03.01