苧麻莖皮環狀RNA表達譜分析

李 富 王延周 嚴 理 朱四元 劉頭明

專題

苧麻莖皮環狀RNA表達譜分析

李 富 王延周 嚴 理 朱四元 劉頭明*

農業農村部麻類生物學與加工重點實驗室 / 中國農業科學院麻類研究所, 湖南長沙 410205

苧麻[(L.) Gaud.]是我國特色的天然纖維作物, 其纖維具有拉伸力強、纖維束長等特點。解析苧麻纖維發育調控機制, 對實現纖維產量和品質性狀遺傳改良具有重要意義。本研究基于高通量測序技術開展了苧麻莖頂部和中部的莖皮組織環狀RNA (circRNA)分析, 在2個組織中共鑒定到5268個苧麻circRNA。比較circRNA的表達水平發現, 78個circRNA在2個組織中呈現差異表達。因苧麻莖中部韌皮纖維處于生長發育期, 而莖頂部韌皮纖維尚未起始生長, 推測這78個circRNA是纖維不同發育時期差異表達基因, 它們可能參與了苧麻的纖維發育調控。研究結果將為解析circRNA調控苧麻纖維生長發育機制奠定基礎。

苧麻; 纖維發育; 環狀RNA; 差異表達

苧麻[(L.) Gaud.]是我國重要的天然纖維作物, 有著悠久的栽培和加工歷史, 其纖維及纖維織品是我國重要的工業原料和傳統的出口創匯產品。中國作為苧麻的主產國, 其種植面積占全世界90%以上, 因此在國際上苧麻又被稱為“中國草”。苧麻纖維是一種韌皮纖維, 具有拉伸力強、纖維長等特點, 其單纖維最長可達55 cm[1], 主要成分為纖維素、半纖維素和木質素。

目前, 苧麻中已有大量可能與纖維發育相關的基因被鑒定, 這為深入解析其纖維形成機制奠定了基礎。基于RACE技術, 多個苧麻纖維素合成酶基因()已被鑒定和克隆[2-4]。木質素作為纖維主要成分之一, 其多個生物合成酶基因, 如肉桂酸4-羥化酶()、香豆酸4-香豆酸:輔酶A連接酶()、肉桂酰輔酶a還原酶基因()也被鑒定和克隆[5-7], 并發現它們的表達水平與苧麻纖維木質素含量顯著相關[8]。此外, 近10年來高通量測序技術快速發展, 也被廣泛應用于苧麻纖維發育機制研究。Liu等[9]分析苧麻轉錄組共獲得36個在韌皮部中高表達的苧麻基因。Chen等[10]比較不同發育時期的苧麻韌皮組織轉錄組表達譜, 共鑒定到780個差異表達的基因, 其中含4個基因、3個擴張蛋白基因、6個木聚糖水解酶基因, 它們很可能與苧麻的纖維發育相關。

非編碼RNA是一類直接發揮催化和調控功能的轉錄本, 包含miRNA、長鏈非編碼RNA (lncRNA)和環狀RNA (circRNA)等[11]。Wang等[12]分析了苧麻纖維在4個不同發育時期的miRNA表達譜, 并在纖維伸長期和次生壁加厚期分別鑒定到150個和148個miRNA, 其中51個miRNA在纖維不同發育期呈現差異表達, 推測非編碼RNA可能參與苧麻的纖維發育。circRNA是一類由mRNA前體經反向可變剪切而來的共價閉合且保守的環狀轉錄本, 根據剪接位點可分為exonic circRNAs、intronic circRNAs、exonic-intronic circRNAs和intergenic circRNAs, 它們大多數具有較低的表達量, 且呈現組織特異性和細胞特異性表達。circRNA已知的功能包括隔離miRNA或蛋白質、轉錄調節和干擾可變剪接, 甚至翻譯產生多肽[13]。植物circRNA主要集中于生長發育及生物和非生物逆境研究[14-19], 如Zhang等[14]在玉米和擬南芥中分別鑒定出2174個和1354個干旱相關的circRNA; Zuo等[15]鑒定出番茄163個冷脅迫響應的circRNA; Wang等[16]在野生型和有義/反義轉基因番茄果實中發現282個顯著差異表達的cicrRNA; Wang等[18]在小麥中發現62個circRNA響應干旱逆境; Gao等[19]在葡萄中鑒定了475個響應寒冷的差異表達circRNA。盡管circRNA被發現廣泛參與了植物的生長調控, 但有關它在植物纖維發育中的功能角色, 當前仍鮮有研究。本研究將開展苧麻纖維發育期的全基因組circRNA表達譜分析, 并篩選差異表達的circRNA, 旨在為理解circRNA在植物纖維發育中的調控角色奠定基礎。

1 材料與方法

1.1 試驗材料和取樣

本試驗以中苧1號為研究材料。2016年, 將中苧1號扦插苗栽種于中國農業科學院麻類研究所沅江田間試驗基地。經切片顯微觀察, 莖中部韌皮纖維處于生長期, 次生壁正在加厚; 而莖頂部韌皮纖維尚未起始生長[10]。因此, 在2018年5月, 當具有兩年齡的苧麻長到約1.5 m高時, 按照Chen等[10]方法, 分別取莖中部(the barks sample collected from the middle, MPS)和頂部(the barks sample collected from the top, TPS)的莖皮組織(圖1)。對3株苧麻單獨取樣, 分別作為3個試驗重復。組織樣品經液氮速凍后置于-80℃低溫保存, 以備后續試驗使用。

MPS和TPS分別指從莖中部和頂部收集的莖皮組織樣品, 其中位于莖頂端的纖維細胞次生壁尚未加厚, 而處于莖中部的纖維細胞次生壁顯著加厚(圖片引自Chen等[10])。

MPS and TPS represent the barks sample collected from the middle and top stems, respectively. This picture displays that the secondary cellular walls (SCWs) of fiber cells from the top of the ramie stems do not initiate growth and those from middle part of the ramie stems are thickening. This figure is cited from the study of Chen et al.[10]

1.2 文庫構建和高通量測序

利用植物RNA提取試劑TRIzol (Invitrogen, CA, USA)提取6個樣品的總RNA。之后對6個RNA樣品單獨構建circRNA測序文庫, 具體流程如下: 使用試劑盒(Ribo-Zero Gold rRNA Removal Kit, Illumina)去除total RNA中的核糖體RNA, 并使用Agilent 2100Bioanalyzer (Agilent Technologies)驗證純化效果; RNaseR反應體系(TruSeq Stranded Total RNA with Ribo-Zero Plant, Illumina, USA)消化線性RNA, 將反應產物純化, 并打斷使其片段化; 以片段化環狀RNA為模板, 使用反轉錄試劑盒(SuperScript III First-Strand Synthesis System for RT-PCR, Invitrogen)先后合成第一鏈cDNA和第二鏈cDNA, 并使用試劑盒(PureLink Quick Gel Extraction and PCR Purification Combo Kit, Invitrogen)純化回收、黏性末端修復、cDNA的3￠末端加上堿基“A”并連接接頭, 最后進行PCR擴增; 構建好的文庫用Agilent2100 Bioanalyzer (Agilent Technologies)和StepOnePlus Real-time PCR System (ABI)質檢, 合格后的文庫則通過Illumina測序平臺(HiSeq2500, Illumina, USA)進行高通量測序, 從而獲得原始的序列reads。

1.3 circRNA預測

對原始序列進行過濾, 主要包括去除接頭污染的序列reads、未知堿基N含量大于5%的reads及低質量的序列reads, reads中超過20%的堿基為質量值低于30的堿基(即堿基錯誤率大于0.032), 從而獲得高質量的clean reads。將clean reads比對到苧麻參考基因組上[20], 然后通過CIRI[21]、find_circ[22]2個軟件預測circRNA, 并且依據circRNA起始、終止位置來整合2個軟件的預測結果。若2個circRNA的起始和終止位置均相近(10個堿基以內), 則將這2個circRNA合并為1個。

1.4 circRNA表達定量及差異表達分析

本研究根據比對至circRNA兩端的junction reads數來對circRNA表達定量[23]。因circRNA是由CIRI和find_circ 2個軟件預測而來, 故就每個circRNA, 我們計算2個軟件注釋的junction reads數的平均值, 將其作為該circRNA最終的junction reads數。采用RPB (junction reads per billion mapped reads, 即比對上基因組的所有reads標準化到十億后跨過backspliced位點的junction reads數目)對各樣品作均一化處理。原始的clean reads序列及其表達定量已被提交到DDBJ/EMBL/GenBank數據庫(序列號位GSE130587)。

利用DEseq2軟件[24]篩選差異表達的circRNA。利用DEseq2軟件計算circRNA在莖頂部和莖中部的表達量差異倍數和值, 通過Benjamini-Hochberg統計方法[25]對值進行校正。對于每個circRNA, 若其校正的值小于0.05, 表達差異大于2倍, 則認為該circRNA在2個組織中存在表達差異。

1.5 熒光定量PCR (qRT-PCR)分析

分別提取莖頂部和莖中部RNA (3個生物學重復, 共6個樣品), 用DNase I (Fermentas, Canada)進行消化處理, 去除其中的DNA。之后, 利用M-MuLV反轉錄試劑盒(Fermentas)合成第1鏈cDNA。使用iTaq Universal SYBR Green super mix試劑盒(Bio-Rad, USA)配置qRT-PCR反應體系, 并在iQ5實時熒光PCR儀上進行PCR。PCR反應程序: 第一步95℃30 s; 第二步40個循環: 95℃15 s, 60 ℃30 s。就每個circRNA, 每個樣品進行5個PCR反應重復。18S核糖體RNA在植物各器官中呈現組成性表達, 因此被用作內參基因[12]。circRNA和內參基因引物見表1。依據Livak的2?ΔΔCT方法[26]計算各circRNA在2個組織中的相對表達水平和標準誤。

表1 用于qRT-PCR分析的苧麻差異表達circRNA和內參基因引物序列

2 結果與分析

2.1 circRNA的鑒定

經測序, 6個樣品共產生大約6.46億個原始序列reads, 經過濾后得到共計4.45億個高質量的clean reads, 序列長度合計達到66.7 Gb (表2)。每個樣品過濾后均有約74.0百萬個clean reads, 且其Q30值均達到96%以上, 說明測序獲得的數量和質量均足以開展下一步的circRNA分析。

基于這些高質量的序列, 本研究在苧麻莖頂部和中部韌皮組織中共鑒定到5268個circRNA, 其中539個位于基因間(intergenic), 而4729個(89.8%)則位于基因內部(intragenic)。對這4729個基因進行GO (gene ontology)功能分類發現, 它們主要歸類到結合(binding)、催化活性(catalytic activity)、細胞過程(cellular process)、代謝過程(metabolic process)、膜(membrane)等功能類別(Q < 0.01)(圖2)。

表2 苧麻6個莖皮組織樣本的circRNA測序數據統計

MPS和TPS分別指從莖中部和頂部收集的莖皮組織樣品; Q30值為序列過濾后的reads中質量值大于30的堿基數占總堿基數的比例。

MPS and TPS represent the barks sample collected from the middle and top stems, respectively; Q30 value indicates the ratio of bases with quality of more than 30 in the clean read.

2.2 circRNA表達譜分析

顯微觀察顯示, 苧麻莖中部韌皮纖維處于生長期, 次生壁正在加厚; 而莖頂部韌皮纖維尚未起始生長(圖1)。比較5268個苧麻circRNA在2個莖韌皮部位中的表達水平發現, 相對于TPS, 在MPS韌皮中有77個circRNA顯著上調表達, 僅1個circRNA表達水平下調(圖3)。因莖中部和頂部的纖維處于不同發育時期, 說明這78個circRNA在2個不同纖維發育時期存在表達差異。在這78個circRNA中, 11個circRNA的表達水平差異倍數達到30倍以上(表3)。其中scaffold13:3497301|3498287和scaffold7:2328081|2328970在MPS中的表達分別上調了42.7倍和42.0倍, 它們分別定位在苧麻基因和中;編碼貝殼杉烯酸氧化酶,則為酪蛋白激酶編碼基因。此外, 在這78個差異表達的circRNA中, 74個(94.9%)位于基因內部, 其中scaffold1:2974233| 2974487被定位在一個knotted-1-like Homeobox基因()中, 其上調表達倍數達到30.6。

為了驗證這些circRNA的差異表達結果, 本研究隨機選取了10個circRNA, 通過qRT-PCR進一步分析它們在2個組織中的表達水平。由圖4可知, 10個circRNA均呈現出差異表達, 且差異表達倍數明顯高于測序分析結果, scaffold6:1851520| 1851960和scaffold30:600099|602644的差異表達倍數甚至達到上千倍。可見, 與高通量測序技術相比, qRT-PCR檢測circRNA具有更高的靈敏度。說明這78個circRNA的差異表達是真實可靠的。

3 討論

纖維廣泛存在于植物各器官中, 具有支撐器官形態、植株直立生長, 及抵抗病原菌攻擊等作用。此外, 植物纖維也是重要的工業原料, 廣泛用于紡織、造紙等工業領域。可見, 不管是對植物本身, 還是對國民經濟, 植物纖維具有非常重要的作用。目前, 有關植物纖維生物合成機制在模式植物擬南芥中研究的比較多, 發現其主要由一系列NAC (NAM, ATAF1/2和CUC2)和MYB (v-myb avian myloblastosis viral oncogene homolog)轉錄因子調控, 它們形成分層次的網絡并逐級調控整個纖維發育過程[27]。蛋白翻譯后修飾如磷酸化、泛素化等也參與植物纖維的發育調控[28-30]。此外, 非編碼RNA也可參與植物的纖維發育調控, 如柑橘可以調控纖維發育控制基因和的表達[31], 而擬南芥則靶向調控基因的表達, 進而抑制纖維發育[32]。

圖中紅色、藍色和灰色的數據點分別代表上調表達、下調表達及無表達差異的circRNA; 橫坐標和縱坐標分別代表某circRNA在TPS和MPS組織中的read數的對數值。縮寫同表2。

Red dots represent transcripts more prevalent in the MPS library, blue dots show those present at a lower frequency in the MPS, and gray dots indicate transcripts that did not change significantly; X and Y axes represent the logarithm of read number for each circRNA in TPS and MPS libraries, respectively. Abbreviations are the same as those given in Table 2.

circRNA是一類重要的非編碼RNA, 廣泛參與花發育、果實成熟、逆境響應等多個生命過程[33]。植物中存在大量的circRNA, 如PlantcircBase數據庫[34]已收錄了40,311個水稻() circRNA、38,938個擬南芥() circRNA、6302個玉米() circRNA、7806個大豆() circRNA和1976個番茄() circRNA。Wang等[35]系統研究了244個玉米RNA-Seq樣本和288個水稻RNA-Seq樣本, 鑒定到38,785個玉米circRNA和63,048個水稻circRNA, 并發現在玉米中有11,206個circRNA響應干旱逆境, 6770個circRNA響應鹽逆境, 而在水稻中有824、6313和5724個circRNA分別響應干旱、鹽和冷逆境。Zhang等[36]首次發現可能與花生種子發育相關的347個差異表達circRNA, 并通過qRT-PCR驗證了其中15個circRNA的表達水平。茉莉酸甲酯(methyl jasmonic acid, MeJA)在植物生長和防御中發揮關鍵作用, 擬南芥種子用MeJA處理后, 經高通量測序鑒定出8588個circRNA, 其中有385個circRNA響應MeJA處理[37]。已有研究表明, circRNA能夠參與調控其宿主基因表達, 例如在水稻中過表達circRNA Os08circ16564降低其宿主基因在葉和花序中的表達[37]。Gao等[19]在低溫脅迫下的葡萄葉片中鑒定了475個差異表達的circRNA, 通過異源過度表達一種源自甘油-3-P-酰基轉移酶的circRNA (Vv-circATS1), 可以增強擬南芥(Arabidopsis thaliana)的耐寒性, 而源自同一序列的線性RNA則不能, 這為植物耐寒提供新見解。

圖4 qRT-PCR分析10個苧麻circRNA在MPS和TPS莖皮中的相對表達水平

Fig. 4 Relative expression level of ten circRNAs stem bark tissues of MPS and TPS by qRT-PCR in ramie

縮寫同表2。Abbreviations are the same as those given in Table 2.

當前對circRNA在植物纖維發育中的功能角色仍有待深入研究。本研究鑒定到78個苧麻circRNA在纖維發育不同時期呈現差異表達, 這將為研究circRNA在苧麻纖維發育中的功能奠定重要基礎。在78個差異表達circRNA中, 有74個circRNA位于基因內部, 占比達94.9%, 這個比例高于苧麻總circRNA中該類型的比例(89.8%), 其表達是否影響相應基因的表達, 目前仍難以確定。例如knotted-1-like Homeobox家族是植物發育中重要的轉錄因子家族, 其多個成員, 如擬南芥、棉花均被發現參與植物的纖維發育調控[38-39]。此外, 在纖維發育過程中, 胞外復合體酶EXO70A1主要負責轉運纖維素合成酶[40], 而磷酸酶SAC1在纖維次生壁加厚過程中具有重要功能[41]。本研究發現,、和分別注釋為擬南芥纖維發育基因、和的同源基因, circRNAscaffold1:2974233|2974487、scaffold23:488065| 489466和scaffold69:125839|129078分別定位到這3個苧麻基因中。因circRNA可通過miRNA海綿功能、干擾可變剪切、結合蛋白等方式調控相應基因表達, 這3個circRNA的上調表達或許會影響到相應的、和表達, 進而可能在纖維發育中發揮功能。因此, 本研究將為我們深入研究這3個circRNA和相應基因的功能及它們的表達調控關系奠定基礎。

4 結論

本研究首次在苧麻中鑒定到5268個circRNA, 并發現其中78個circRNA在纖維發育的2個不同時期呈現差異表達。這些circRNA的鑒定為研究苧麻生長發育, 尤其是纖維的生長發育調控功能提供重要基礎。

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Characterization of the expression profiling of circRNAs in the barks of stems in ramie

LI Fu, WANG Yan-Zhou, YAN Li, ZHU Si-Yuan, and LIU Tou-Ming*

Key Laboratory of Biological and Processing for Bast Fiber Crops, Ministry of Agriculture and Rural Affairs / Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, Hunan, China

Ramie [(L.) Gaud.] is a special natural fiber crops in China, and its fiber has many excellent characteristics, including long strands and well tensile strength. Elucidation of the mechanism for fiber formation will be helpful for the improvement of fiber yield and quality in ramie. In this study, the expressed analysis of circular RNA (circRNA) for the tissues of barks from the top stems and middle stems were performed by Illumina sequencing, respectively. The total of 5268 circRNAs were identified. Among these circRNAs, 78 showed differential expression between two examined tissues. Previous cytological observation suggested that the secondary cellular walls (SCWs) of fiber cells from the top of the ramie stems did not initiate growth and those from middle part of the ramie stems were thickening. Therefore, we speculated that these 78 differentially expressed circRNAs were potentially involved in the fiber development in ramie. The results provide an important basis for understanding the role of circRNA in the regulation of fiber development.

ramie; fiber development; circRNA; differential expression

10.3724/SP.J.1006.2021.04042

本研究由國家自然科學基金項目(31871678)和中國農業科學院科技創新工程項目(CAAS-ASTIP-IBFC)資助。

This study was supported by the National Natural Science Foundation of China (31871678) and the Agricultural Science and Technology Innovation Program of China (CAAS-ASTIP-IBFC).

劉頭明, E-mail: liutouming@caas.cn

E-mail: 704510340@qq.com

2020-02-24;

2020-07-02;

2020-07-16.

URL: https://kns.cnki.net/kcms/detail/11.1809.S.20200715.1817.006.html