長鏈非編碼RNA (LncRNA)在印度梨形孢促進(jìn)大麥根部生長發(fā)育中的調(diào)控作用

郭楠楠 劉天策 史 碩 胡心亭 牛亞丹 李 亮

長鏈非編碼RNA (LncRNA)在印度梨形孢促進(jìn)大麥根部生長發(fā)育中的調(diào)控作用

郭楠楠 劉天策 史 碩 胡心亭 牛亞丹 李 亮*

河北工業(yè)大學(xué)化工學(xué)院, 天津 300130

印度梨形孢定殖植物會促進(jìn)植物生物產(chǎn)量提高, 其分子機(jī)制有待深入挖掘。LncRNA是一類長鏈非編碼RNA, 在植物生長發(fā)育過程中具有重要調(diào)控作用。然而, 目前我們還不清楚大麥中LncRNA是否對印度梨形孢的定殖有響應(yīng)。本研究發(fā)現(xiàn), 印度梨形孢定殖大麥會誘導(dǎo)大麥根系迅速發(fā)育, 促進(jìn)較多根分枝。采用全基因組高通量測序RNA-seq和生物信息學(xué)方法鑒定LncRNA, 發(fā)現(xiàn)在定殖后3 d和7 d分別有752個和932個差異表達(dá)的LncRNA, 7 d相對于3 d有70個差異表達(dá)的LncRNA。其中在定殖后3 d有375個LncRNA表達(dá)上調(diào), 377個LncRNA表達(dá)下調(diào); 在定殖后7 d有459個LncRNA表達(dá)上調(diào), 473個LncRNA表達(dá)下調(diào); 7 d相對于3 d組中, 有39個LncRNA表達(dá)上調(diào), 31個LncRNA表達(dá)下調(diào)。qPCR驗(yàn)證LncRNA的表達(dá)與RNA-seq結(jié)果一致。GO和KEGG分析表明, 在定殖大麥促生過程中, 部分LncRNA參與了激素信號途徑的轉(zhuǎn)錄調(diào)控。該工作對于進(jìn)一步理解LncRNA與靶基因的相互作用以及其對靶基因的調(diào)控功能提供了新的理論基礎(chǔ)和實(shí)驗(yàn)依據(jù), 并以LncRNA為靶點(diǎn), 進(jìn)行作物性狀改良提供新的思路和方向。

LncRNA; 大麥; 印度梨形孢; RNA-seq; 轉(zhuǎn)錄因子; 細(xì)胞周期

研究人員曾經(jīng)認(rèn)為長鏈非編碼RNA (LncRNA)是轉(zhuǎn)錄過程中產(chǎn)生的“無用物”, 最近發(fā)現(xiàn)其在植物的諸多調(diào)控過程中發(fā)揮重要作用[1]。盡管大多數(shù)LncRNA功能未知, 但已鑒定的LncRNA表現(xiàn)出組織特異性表達(dá)并參與蛋白定位以及調(diào)控相關(guān)疾病的特點(diǎn)[2-3]。順式(或反式) LncRNA具有調(diào)節(jié)蛋白質(zhì)活性或定位[4-5]的作用, 并可作為亞細(xì)胞結(jié)構(gòu)的組織框架[2-3,6]。此外, 某些LncRNA可作為競爭性內(nèi)源RNA (ceRNA)與小分子RNA (microRNA)配對[7-9]。

目前已對部分植物在遭受冷、熱、干旱、鹽和氮等一系列脅迫反應(yīng)中的LncRNA進(jìn)行了鑒定, 如毛果楊[10]、蒺藜苜蓿[11]、棉花[12]、玉米[13]和小麥[14-15]等植物。此外, 在對擬南芥的研究中發(fā)現(xiàn), 冷誘導(dǎo)的LncRNA COOLAIR在春化早期起作用, 基因3′末端的反義轉(zhuǎn)錄機(jī)制可能是以條件/階段依賴的方式調(diào)控基因轉(zhuǎn)錄, LncRNA COLDAIR可調(diào)節(jié)春化介導(dǎo)的表觀遺傳沉默從而應(yīng)對冷脅迫[16-17]; LncRNA LDMAR在雜交稻中起著調(diào)節(jié)光周期敏感雄性不育的作用[18]; LncRNA DRIR能正向調(diào)控擬南芥對干旱和鹽脅迫的響應(yīng)[19]。另外, Zou等[20]和Karlik等[21]在大麥全基因組范圍內(nèi)發(fā)現(xiàn)了大約8000個LncRNA,并揭示了它們在過量硼處理下的表達(dá)模式。此外, 與LncRNA共表達(dá)編碼轉(zhuǎn)錄本的功能注釋揭示了離子轉(zhuǎn)運(yùn)、定位建立和葉對刺激響應(yīng)的分子功能[22]。LncRNA HvCesA6調(diào)控大麥細(xì)胞壁的合成[23]?；贚ncRNA-mRNA相互作用表明, LncRNA介導(dǎo)的調(diào)控絲氨酸/蘇氨酸蛋白激酶SMG1信號轉(zhuǎn)導(dǎo)途徑可能與野生大麥XZ5的耐旱性有關(guān)[24]。Karlik等[25]報道了鹽脅迫下大麥通過抑制LncRNAAK370814的表達(dá)反向促進(jìn)基因表達(dá), 并進(jìn)一步明確了LncRNA-AK363461和LncRNAAAK370506在細(xì)胞中的定位。這些發(fā)現(xiàn)增進(jìn)了我們對LncRNA作為抗旱重要調(diào)節(jié)因子的認(rèn)識。然而, LncRNA在更廣泛的環(huán)境條件下的生物學(xué)功能還需深入探索。

內(nèi)生擔(dān)子菌印度梨形孢()屬蠟殼耳目, 梨形孢屬[26]。與大麥、擬南芥、小麥、玉米、煙草、番茄[2,27-31]等多種植物形成了互惠菌根共生關(guān)系。具有促進(jìn)植物生長、增強(qiáng)抗逆性和抗病性、促進(jìn)營養(yǎng)物質(zhì)和生物活性物質(zhì)積累等多種功能[32-37]。分子生物學(xué)研究表明, 接種可誘導(dǎo)與激素信號轉(zhuǎn)導(dǎo)、細(xì)胞壁代謝、碳水化合物運(yùn)輸代謝和根系形成等相關(guān)基因表達(dá)[38-39]。然而, 在促進(jìn)大麥生長中, LncRNA的響應(yīng)及調(diào)控機(jī)制仍未見報道, 需進(jìn)行深入探索。

1 材料與方法

1.1 植株生長

將春大麥品種福大麥1號(162粒)用70%乙醇表面殺菌3 min, 然后在次氯酸鈉(5%活性氯)中殺菌20 min。在無菌條件下, 用無菌水(pH 3.0)清洗1次, 無菌蒸餾水沖洗6次。將滅菌種子放在1/2 MS培養(yǎng)基的方形培養(yǎng)皿中, 每個培養(yǎng)皿中種6粒種子, 每個處理組(對照組即Mock、印度梨形孢定殖后3 d、印度梨形孢定殖后7 d)進(jìn)行3個重復(fù)。在20℃的培養(yǎng)箱中培養(yǎng), 條件如下: 光照8 h (熒光冷白, Toshiba FL40SSW/37, 180 μmol m–2s–1光子通量密度)/黑暗16 h, 22℃/18℃, 相對濕度60%。為了保證所有根系都能在培養(yǎng)基表面生長, 將含有種子的培養(yǎng)皿垂直放置。培養(yǎng)結(jié)束后, 挑選每個處理組長勢良好的大麥各36顆, 對其根部進(jìn)行取樣, 樣品送天津諾禾致源生物信息科技有限公司進(jìn)行測序。

1.2 植物根系接種印度梨形孢

在CM培養(yǎng)基上生長3～4周, 可制備孢子懸浮液。在CM培養(yǎng)基中加入0.05%吐溫-20的滅菌水, 用涂布棒輕輕刮擦平板表面, 收集孢子, 懸浮液通過miracloth (中國鼎國)過濾以除去菌絲體。將懸浮液在1359 ×下離心7 min收集孢子。用滅菌的吐溫水清洗孢子3次。顯微鏡下, 血球計數(shù)板測定孢子密度。將孢子用無菌吐溫水稀釋濃度至105個孢子mL–1。接種時, 用移液槍將3 mL孢子懸浮液滴到方形培養(yǎng)皿中的植物根表面。

1.3 RNA提取及測序定量與鑒定

用TRIzol (Invitgen, #15596-018)法將對照組和處理組的根(0.5 g)在液氮中研磨成粉末。在1%瓊脂糖凝膠上監(jiān)測RNA降解和污染。分別使用NanoPhotometer分光光度計和Qubit 2.0 Flurometer中的Qubit RNA試劑盒檢測RNA純度與濃度。純化的RNA用Revert-Aid第1鏈cDNA合成試劑盒進(jìn)行反轉(zhuǎn)錄。根據(jù)制造商的說明, 使用Ribo-Zeror RNA Removal Kit (Epicentre, BioTechnoLogies)和NEB Next RULtraTMRNA Library Prep Kit for Illumina (New EngLand BioLabs)構(gòu)建RNA-Seq文庫。根據(jù)末端配對法, 使用Illumina HiSeq 2000對得到的文庫進(jìn)行測序。每個樣本設(shè)置3個生物學(xué)重復(fù)。

1.4 差異表達(dá)基因的GO和KEGG富集分析

GO (Gene Ontology)是描述基因功能的綜合性數(shù)據(jù)庫, 可分為分子功能(molecular function)、生物過程(biological process)和細(xì)胞組成(cellular component) 3個部分。利用clusterProfiler R軟件包與GO網(wǎng)站(http://geneontology.org/)對差異表達(dá)的LncRNA進(jìn)行GO富集分析。KEGG (Kyoto Encyclopedia of Genes and Genomes)是一種數(shù)據(jù)庫資源, 用于從分子水平信息, 尤其是基因組測序等高通量實(shí)驗(yàn)技術(shù)產(chǎn)生的大規(guī)模分子數(shù)據(jù)集(http://www.genome.jp/kegg/)了解生物系統(tǒng)(如細(xì)胞、生物體和生態(tài)系統(tǒng))的高級功能和實(shí)用性。使用clusterProfiler R軟件包測試KEGG通路中差異表達(dá)的LncRNA的統(tǒng)計富集情況。GO富集和KEGG通路富集以-value或adj小于0.05為顯著富集。

1.5 實(shí)時定量聚合酶鏈反應(yīng)(qRT-PCR)分析

分別用對照和處理組的總RNA (1 μg)與M-MLV Reverse轉(zhuǎn)錄酶來制備cDNA。經(jīng)DNase I處理后, 將cDNA作為qRT-PCR的模板, 使用LncRNA特異性引物和靶mRNA特異性引物對所選擇的LncRNA和mRNA進(jìn)行定量分析。定量RT-PCR檢測各基因的表達(dá)水平(SYBR綠色熒光法測定)。qPCR實(shí)驗(yàn)在Light Cyker96快速實(shí)時PCR系統(tǒng)(Roche)上進(jìn)行, 該反應(yīng)溶液含有2×ULtra SYBR 混合物10 μL, cDNA模板100 ng , 正反向引物10 μmol L–1。以HvUBIQUTIN為對照, 進(jìn)行3次重復(fù)。擴(kuò)增程序按以下步驟進(jìn)行: 初始活化步驟在95℃下進(jìn)行5 min, 然后進(jìn)行30個循環(huán)(95℃ 20 s, 56℃ 35 s, 72℃ 35 s, 65℃ 20 s)。在每個循環(huán)結(jié)束時, 分別測定熔融曲線, 以保證單個PCR產(chǎn)物的擴(kuò)增。

2 結(jié)果與分析

2.1 P. indica的定殖提高了大麥根的產(chǎn)量

大麥幼苗在處理的第3天和第7天的根長均比對照組顯著增長(圖1-A, B)。對3 d和7 d處理組與Mock組相比, 根長度分別增加了75%和58%。此外, 根在定殖7 d后分枝顯著, 根數(shù)在3 d和7 d比Mock組分別增加了25%和42.85% (圖1-C, D)。該結(jié)果表明,定殖能促進(jìn)大麥根系生長和根數(shù)增加。

2.2 大麥根系對P.indica定殖反應(yīng)的差異表達(dá)的LncRNA譜

為揭示大麥LncRNA對定殖的分子響應(yīng)機(jī)制, 從12個大麥根部樣本中提取RNA, 并進(jìn)行全轉(zhuǎn)錄組序列測定。LncRNA鑒定的高通量測序流程和結(jié)果見圖2。每個樣本的RNA序列產(chǎn)生了1.042～1.285億次raw reads和1.038～1.252億次clean reads。Raw reads提交至NCBI (PRJNA71930)。利用Hisat將組裝好的clean reads映射到大麥基因組上, 所有樣本的平均映射率為74.13% (附表1)。利用RSeQC測定RNA-seq中LncRNA的飽和度見附表2。

圖1 大麥在P. indica定殖后3 d和7 d形態(tài)、根長與根數(shù)統(tǒng)計結(jié)果圖

A: 處理組與對照組在3 d的根長和根數(shù), 標(biāo)尺為 1.0 cm; B: 處理組與對照組在3 d根的分枝情況, 標(biāo)尺為1.0 cm; C: 處理組與對照組在3 d和7 d的根長統(tǒng)計; D: 處理組與對照組在3 d和7 d的根數(shù)統(tǒng)計。進(jìn)行3個生物學(xué)重復(fù)實(shí)驗(yàn)和統(tǒng)計分析。

A: root length and root number were compared between treatment and Mock at 3 days. Bar: 1.0 cm; B: branched roots were compared between treatment and Mock at 3 days. Bar: 1.0 cm; C: chart showing the statistical results of the root length of barley at 3 days and 7 days; D: chart showing the statistical results of the root number of barley at 3 days and 7 days.The experiments and statistical analysis were performed in three biological replicates.

圖2 LncRNA鑒定的高通量測序流程圖

層次聚類熱圖(圖3)表明3個處理組(即3 d vs Mock、7 d vs Mock和7 d vs 3 d)的差異表達(dá)的LncRNA表達(dá)豐度不同, 說明這些基因位于不同的代謝途徑及信號通路中。由圖4可知, 在3 d vs Mock組中, 共有752個差異表達(dá)的LncRNA, 其中, 有375個LncRNA表達(dá)上調(diào), 377個LncRNA表達(dá)下調(diào); 在7 d vs Mock組中, 共有932個差異表達(dá)的LncRNA, 其中上調(diào)表達(dá)的LncRNA有459個, 下調(diào)表達(dá)的LncRNA有473個; 在7 d vs 3 d組中, 共有70個差異表達(dá)的LncRNA, 其中39個LncRNA表達(dá)上調(diào), 31個LncRNA表達(dá)下調(diào)。

采用qRT-PCR對RNA-seq測序結(jié)果進(jìn)行驗(yàn)證。使用特異性引物對隨機(jī)選擇的6個基因片段進(jìn)行定量分析(表1)。結(jié)果表明, XLOC_070335、XLOC_318641和XLOC_067560這3個基因表達(dá)上調(diào); XLOC_119623、XLOC_322519和XLOC_206343基因表達(dá)下調(diào), 這6個基因片段的表達(dá)與測序分析結(jié)果一致(圖5), 說明測序結(jié)果是可信的。

2.3 大麥根系對P. indica定殖反應(yīng)的差異表達(dá)的LncRNA功能分析

在生物體內(nèi), 不同基因相互作用, 從而表現(xiàn)出生物學(xué)功能。通過pathway顯著性富集可以探尋差異表達(dá)基因參與的最主要的生化代謝途徑和信號轉(zhuǎn)導(dǎo)途徑。為了進(jìn)一步研究這些LncRNA的功能, 我們對3個處理組中顯著上調(diào)(附表3～附表5)和下調(diào)(附表6～附表8)的LncRNA進(jìn)行了GO富集分析, 并繪制了GO富集柱狀圖(圖6)。結(jié)果表明, 這些差異表達(dá)的LncRNA參與了生物學(xué)過程(BP)、細(xì)胞成分(CC)和分子功能(MF)。在3 d vs Mock組中對比顯示, 在BP中, 差異表達(dá)的LncRNA在“代謝途徑”和“單一生物代謝途徑”中所占比例較高。在CC中, 占比高的部分為“細(xì)胞核”, 在MF中, 差異表達(dá)的LncRNA主要參與“催化活性”、“分子功能”和“結(jié)合”等過程。在7 d vs Mock組中對比顯示, 在BP中, 差異表達(dá)的LncRNA在“代謝途徑”、“單一生物代謝途徑”與“蛋白質(zhì)代謝途徑”中所占比例較高, 在CC中, 占比高的部分為“細(xì)胞核”, 在MF中, 差異表達(dá)的LncRNA主要參與“催化活性”、“分子功能”、“結(jié)合”和“離子結(jié)合”等過程。在7 d vs 3 d組中對比顯示, 在BP中, 差異表達(dá)的LncRNA在“防御反應(yīng)”和“單一生物碳水化合物代謝途徑”中所占比例較高, 在CC中, 統(tǒng)計到差異表達(dá)的LncRNA在“Smc5-Smc6絡(luò)合物”、“U2型剪接體復(fù)合體”、“U2型mRNA釋放后剪接體”、“mRNA釋放后剪接體復(fù)合體”和“H/ACA RNP復(fù)合體”中富集, 在MF中, 差異表達(dá)的LncRNA主要參與“陽離子結(jié)合”與“過渡金屬離子結(jié)合”等過程。

圖3 差異表達(dá)的LncRNA的層次聚類熱圖

橫坐標(biāo)為樣本名稱, 縱坐標(biāo)為差異表達(dá)的LncRNA, 左側(cè)根據(jù)表達(dá)相似程度對基因進(jìn)行聚類, 上方根據(jù)表達(dá)譜的相似程度對每個樣本進(jìn)行聚類, 由藍(lán)至紅表達(dá)量逐漸上調(diào), 數(shù)字為均一化后的相對表達(dá)量。

The abscissa is the sample, the ordinate is the differentially expressed LncRNA. On the left side, the genes are clustered according to the degree of similarity of expression, and the upper part of the sample is clustered according to the degree of similarity of the expression profile. The relative expression level is gradually up-regulated from blue to red, and the number is the relative expression after standardization.

圖4 差異表達(dá)LncRNA火山圖

A: 3 d vs Mock比較組中差異表達(dá)的LncRNA; B: 7 d vs Mock 比較組中差異表達(dá)的LncRNA; C: 7 d vs 3 d比較組中差異表達(dá)的LncRNA。橫坐標(biāo)表示基因在不同樣本或比較組合間的表達(dá)倍數(shù)變化(log2(Fold Change)), 橫坐標(biāo)的絕對值越大表明兩個比較組合之間的表達(dá)變化倍數(shù)越大; 縱坐標(biāo)表示表達(dá)差異的顯著性水平。表達(dá)上調(diào)基因用紅色點(diǎn)表示, 下調(diào)基因用綠色點(diǎn)表示, 藍(lán)色點(diǎn)為未發(fā)生顯著變化的基因(adj< 0.05) 。

A: volcano map of differentially expressed LncRNA in 3 days vs Mock comparison group; B: volcano map of differentially expressed LncRNA in 7 days vs Mock comparison group; C: volcano map of differentially expressed LncRNA in 7 days vs 3 days comparison group. The abscissa indicates the multiple change of gene expression between different samples or comparison combinations (log2(Fold Change)). The larger the absolute value of abscissa indicates the greater the multiple of expression change between the two comparison combinations; the ordinate indicates the significant level of expression difference. The up-regulated genes are represented by red dots, the down-regulated genes are represented by green dots, and the blue dots are genes that have not changed significantly (adj< 0.05).

表1 用于LncRNA差異表達(dá)鑒定的引物

圖5 qRT-PCR鑒定LncRNA的差異表達(dá)

A: LncRNA_XLOC_070335、XLOC_318641和XLOC_067560基因表達(dá)上調(diào); B: LncRNA_XLOC_119623、XLOC_322519和XLOC_206343基因表達(dá)下調(diào)。qRT-PCR實(shí)驗(yàn)和數(shù)據(jù)分析進(jìn)行3個生物學(xué)重復(fù)。*顯著差異(< 0.05)。

A: LncRNA_XLOC_070335, XLOC_318641, and XLOC_067560 were up-regulated; B: LncRNA_XLOC_119623, XLOC_322519, and XLOC_206343 were down-regulated. The experiments of qRT-PCR and the data analysis were performed in three biological replicates. *:< 0.05.

KEGG通路分析采用KEGG API和R程序包Pathview進(jìn)行。圖7總結(jié)了3個對照組在KEGG途徑中富集的差異表達(dá)的LncRNA。在3 d vs Mock組中, 上調(diào)基因在“酪氨酸代謝”、“次級代謝產(chǎn)物生物合成”、“氧化磷酸化”、“苯丙烷生物合成”、“苯丙氨酸代謝”途徑中富集; 而下調(diào)基因在“次級代謝產(chǎn)物生物合成”、“苯丙烷生物合成”、“苯丙氨酸代謝”、“氧化磷酸化”途徑中富集。在7 d vs Mock組中, 上調(diào)基因在“酪氨酸代謝”、“苯丙烷生物合成”、“苯丙氨酸代謝”、“氧化磷酸化”、“代謝途徑”、“次級代謝產(chǎn)物生物合成”途徑中富集; 下調(diào)基因在“苯丙烷生物合成”、“苯丙氨酸代謝”、“氧化磷酸化”、“代謝途徑”、“次級代謝產(chǎn)物生物合成”途徑中富集; 在7 d vs 3 d組中, 上調(diào)基因在“淀粉和蔗糖代謝”、“代謝途徑”、“次級代謝產(chǎn)物生物合成”途徑中富集; 下調(diào)基因在“代謝途徑”、“次級代謝產(chǎn)物生物合成”途徑中富集。

3 討論

大麥?zhǔn)鞘澜缟戏N植最廣泛、消費(fèi)最多的糧食作物之一, 增加大麥產(chǎn)量是解決人口增長和氣候變化問題的重要途徑之一。參與植物代謝途徑的相關(guān)LncRNA基因是影響產(chǎn)量的關(guān)鍵因素, 對植物適應(yīng)環(huán)境脅迫具有重要調(diào)控作用。因此, 本研究對大麥進(jìn)行全轉(zhuǎn)錄組測序旨在揭示LncRNA的潛在調(diào)控機(jī)制。作物與有益內(nèi)生真菌的互作是一種自然而有效的增產(chǎn)方法?？梢蕴岣叨喾N植物的生物產(chǎn)量, 并可誘導(dǎo)植物產(chǎn)生局部和系統(tǒng)抗性[28,36,40]。關(guān)于該真菌和植物的相互作用, 有從轉(zhuǎn)錄組進(jìn)行的研究分析[41-42]。然而, 到目前為止, 還沒有從LncRNA角度揭示對大麥的促生機(jī)制。

為了揭示定殖對大麥促進(jìn)生長的分子機(jī)制, 我們進(jìn)行了高通量RNA-seq分析。共鑒定出22,0761個LncRNA的轉(zhuǎn)錄本。通過KEGG分析植物激素信號轉(zhuǎn)導(dǎo)途徑, 發(fā)現(xiàn)參與生長素途徑的基因(HORVU4Hr1G016160、HORVU2Hr1G109650、HORVU7Hr1G017790)表達(dá)上調(diào)(圖8)。這與前期實(shí)驗(yàn)結(jié)果定殖促進(jìn)了根系的形成與生長相吻合(圖1), 而TCNS_00342393、TCNS_00211320、TCNS_00342393等LncRNA與生長素合成途徑的基因均呈正相關(guān); 此外, 編碼DELLA蛋白的基因(HORVU2Hr1G016120、HORVU3Hr1G088780)表達(dá)下調(diào)。在赤霉素合成途徑中, DELLA蛋白在信號轉(zhuǎn)導(dǎo)中起著抑制子的作用, 這說明定殖能抑制DELLA 蛋白, 從而促進(jìn)赤霉素合成途徑。而LncRNA 如TCONS_00208317、TCONS_00237842、TCONS_00155869、TCONS_00555493均下調(diào)轉(zhuǎn)錄, 并與靶基因DELLA蛋白表達(dá)呈正相關(guān)。以上結(jié)果表明定殖大麥過程中, LncRNA參與了植物激素信號轉(zhuǎn)導(dǎo)途徑關(guān)鍵基因的表達(dá)調(diào)控, 且主要以正向調(diào)控為主。

圖6 GO富集柱狀圖

A: 3 d vs Mock組中GO 的富集; B: 7 d vs Mock組中GO的富集; C: 7 d vs 3 d組中GO的富集; 橫坐標(biāo)表示GO條目名稱, 分為3類(BP生物學(xué)過程, CC細(xì)胞組分, MF分子功能)用不同條框區(qū)分, 縱坐標(biāo)為GO條目富集的基因數(shù)。

A: GO enrichment in 3 days vs Mock group; B: GO enrichment in 7 days vs Mock group; C: GO enrichment in 7 days vs 3 days group. The abscissa denotes the name of GO entry, which is divided into three categories by box (BP biological process, CC cell component, MF molecular function), distinguished by different frames, and the ordinate is the number of genes enriched by GO entry.

(圖7)

圖中縱坐標(biāo)代表不同的通路, 橫坐標(biāo)代表相應(yīng)通路顯著差異表達(dá)基因占該通路所有基因的比例。圓圈大小代表富集在相應(yīng)通路中的基因數(shù)目, 圓圈越大, 代表富集在該通路中的基因越多。顏色代表富集顯著性, 越接近黑色, 代表越顯著。

The ordinate represents different pathways, and the abscissa represents the proportion of differentially expressed genes in the corresponding pathway to all genes in the pathway. The circle size represents the number of genes enriched in the corresponding pathway, and the larger the circle, the more genes are enriched in the pathway. Color represents enrichment significance, and the closer it is to be black, the more significant it is.

圖8 植物激素信號轉(zhuǎn)導(dǎo)途徑

4 結(jié)論

研究發(fā)現(xiàn)定殖后大麥根系生長加快。利用高通量RNA-seq和生物信息學(xué)分析和根部LncRNA全基因組鑒定表明在定殖后3 d和7 d分別有752個和932個差異表達(dá)的LncRNA, 7 d vs 3 d有70個差異表達(dá)的LncRNA。qPCR驗(yàn)證RNA-seq中關(guān)于LncRNA數(shù)據(jù)的有效性。GO及KEEG分析表明, 部分LncRNA參與了大麥根的形成和生長過程中激素信號途徑的轉(zhuǎn)錄調(diào)控, 這些LncRNA可能通過反式作用調(diào)節(jié)靶基因, 也可能作為ceRNAs與某些miRNAs競爭。

附表 請見網(wǎng)絡(luò)版: 1) 本刊網(wǎng)站http://zwxb.china crops.org/; 2) 中國知網(wǎng)http://www.cnki.net/; 3) 萬方數(shù)據(jù)http://c.wanfangdata.com.cn/Periodical-zuowxb.aspx。

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Regulation of long non-coding RNA (LncRNA) in barley roots in response tocolonization

GUO Nan-Nan, LIU Tian-Ce, SHI Shuo, HU Xin-Ting, NIU Ya-Dan, and LI Liang*

College of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China

The molecular mechanism of biomass enhancement byin colonization plants needs to be further explored. LncRNA is a kind of long-chain non-coding RNA, which plays an important role in the regulation of plant growth and development. However, it remains unclear whether barley LncRNAs are responsive tocolonization. It was found that barley roots exhibited fast development and large roots branched aftercolonization. Genome-wide high throughput RNA-seq and bioinformatical analysis showed that 752 and 932 differentially expressed LncRNAs were detected in responsive toat 3-day and 7-day after colonization, respectively. And 70 differentially expressed LncRNAs were found at 7-day compared to 3-day. Among these, 375 were up-regulated and 377 were down-regulated after 3 days’ colonization, and 459 were up-regulated and 473 were down-regulated after 7 days’ colonization, 39 were up-regulated and 31 were down-regulated in 7-day to 3-day comparison group. The qPCR results verified the validity of LncRNAs data in RNA-seq. GO and KEGG analysis indicated that a few LncRNAs might be involved in the molecular functions, cellular components, and biological processes uponcolonization. This study provides a new theoretical basis and experimental basis for further understanding of the interaction between LncRNAs and coding sequences and regulatory functional networks, and provides new ideas and directions for crop shape improvement based on LncRNAs.

LncRNA; barley;; RNA-seq; transcription factor; cell cycle

2021-04-13;

2021-10-19;

2021-11-02.

10.3724/SP.J.1006.2022.11043

通信作者(Corresponding author):李亮, E-mail: liangli@hebut.edu.cn

E-mail: 15033355861@163.com

本研究由國家自然科學(xué)基金項(xiàng)目(31801948), 河北省重點(diǎn)研發(fā)計劃項(xiàng)目(19226505D)和河北省自然科學(xué)基金項(xiàng)目(C2021202005)資助。

This study was supported by National Natural Science Foundation of China (31801948), the Key Research & Development Projects in Hebei Pro-vince (19226505D), and the Natural Science Foundation of Hebei Province (C2021202005).

URL: https://kns.cnki.net/kcms/detail/11.1809.S.20211101.1150.006.html