活性炭促進(jìn)玉米幼胚離體培養(yǎng)幼苗生長(zhǎng)與發(fā)育的轉(zhuǎn)錄組分析

王金萍孫果忠王海波河北農(nóng)業(yè)大學(xué)農(nóng)學(xué)院, 河北保定 0700;河北省農(nóng)林科學(xué)院遺傳生理研究所/河北省植物轉(zhuǎn)基因中心, 河北石家莊 05005

活性炭促進(jìn)玉米幼胚離體培養(yǎng)幼苗生長(zhǎng)與發(fā)育的轉(zhuǎn)錄組分析

王金萍1,2孫果忠2,*王海波2,*
1河北農(nóng)業(yè)大學(xué)農(nóng)學(xué)院, 河北保定 071001;2河北省農(nóng)林科學(xué)院遺傳生理研究所/河北省植物轉(zhuǎn)基因中心, 河北石家莊 050051

以玉米自交系昌7-2為試材, 比較了14 d齡幼胚在MS和MSA (MS加活性炭)培養(yǎng)基上離體培養(yǎng)9 d的幼苗生長(zhǎng)情況, 并利用轉(zhuǎn)錄組測(cè)序技術(shù)分析了2種培養(yǎng)基上的玉米幼苗地上部和地下部的基因表達(dá)差異。結(jié)果表明, 活性炭可以顯著促進(jìn)玉米幼苗的生長(zhǎng)與發(fā)育?；钚蕴恐饕绊懙叵虏炕虮磉_(dá)。加入活性炭后, 幼苗地下部表達(dá)上調(diào)的基因有1612個(gè), 下調(diào)的基因有530個(gè); 幼苗地上部表達(dá)上調(diào)的基因有69個(gè), 下調(diào)的基因有78個(gè)。GO功能顯著性分析表明, 地下部差異表達(dá)基因(DEGs)主要涉及DNA包裝、DNA包裝復(fù)合體和水解酶活性, 地上部DEGs主要涉及脂類代謝、胞外區(qū)和過(guò)氧化物酶活性。KEGG富集分析表明, 地下部 DEGs主要涉及能量、碳水化合物、脂類和氨基酸代謝, 以及細(xì)胞周期和植物激素轉(zhuǎn)導(dǎo)途徑; 地上部 DEGs主要涉及泛醌和其他萜-醌類物質(zhì)合成途徑?；钚蕴看龠M(jìn)細(xì)胞周期途徑中的關(guān)鍵基因(CYC、CDH1、MCM3、PCNA2和BUBR1)、生長(zhǎng)素信號(hào)傳導(dǎo)基因(Aux/IAA)以及細(xì)胞色素基因(CYP450)顯著上調(diào)表達(dá)。利用實(shí)時(shí)熒光定量PCR (qRT-PCR)驗(yàn)證了10個(gè)DEGs的表達(dá)模式與轉(zhuǎn)錄組測(cè)序結(jié)果一致, 證實(shí)了轉(zhuǎn)錄組數(shù)據(jù)與分析的可靠性。

玉米; 幼胚; 活性炭; 轉(zhuǎn)錄組

利用幼胚離體培養(yǎng)直接發(fā)育成苗可以節(jié)省種子發(fā)育成熟的耗時(shí), 顯著縮短植物發(fā)育周期, 有助于加快育種進(jìn)程[1], 建立以幼胚培養(yǎng)為關(guān)鍵步驟的植物快速加代技術(shù)對(duì)促進(jìn)植物育種和遺傳學(xué)研究具有重要意義[2-4]。在適宜條件下, 種子膨脹吸水, 預(yù)存的代謝系統(tǒng)重新活化, 細(xì)胞開(kāi)始伸長(zhǎng)、分裂至胚根突破種皮完成萌發(fā), 隨后進(jìn)入幼苗形態(tài)建成階段[5]。種子萌發(fā)伴隨著一系列有序的生理和形態(tài)發(fā)生過(guò)程,包括呼吸作用加強(qiáng)、能量和物質(zhì)代謝的激活、DNA修復(fù)和蛋白質(zhì)的合成等[6]。種子中貯藏mRNA的翻譯對(duì)種子萌發(fā)非常關(guān)鍵, 抑制翻譯過(guò)程, 則萌發(fā)停止[7-9]。種子萌發(fā)過(guò)程還受植物激素的調(diào)控, 如赤霉素(GA)和脫落酸(ABA)。ABA主要在種子萌發(fā)早期階段起抑制作用, 萌發(fā)條件適宜時(shí), ABA逐漸減少,萌發(fā)順利進(jìn)行[10-12]。GA促進(jìn)種子萌發(fā)主要在胚根突破種皮階段, 使β-糖苷酶、膨脹素基因的表達(dá)上調(diào)[13]。

活性炭(active carbon, AC)是植物微繁殖、組織培養(yǎng)、體細(xì)胞胚發(fā)生和胚胎培養(yǎng)等過(guò)程中常用的添加物[14-16]。一般認(rèn)為 AC通過(guò)其吸附功能和創(chuàng)造培養(yǎng)基的暗環(huán)境, 影響離體培養(yǎng)的效果。AC吸附功能很強(qiáng), 可以吸附培養(yǎng)基中的有害物質(zhì), 如瓊脂中的雜質(zhì)、蔗糖高壓消毒過(guò)程中產(chǎn)生的 5-羥甲基糠醛(5-hydroxymethy-lfurfural)[14]、離體培養(yǎng)過(guò)程中產(chǎn)生的有毒代謝物、酚醛類分泌物[17-19]、生長(zhǎng)調(diào)節(jié)物質(zhì)[20-21]、金屬離子[22]等。強(qiáng)光對(duì)生長(zhǎng)素(如IAA和IBA)具有破壞作用, AC可通過(guò)創(chuàng)造培養(yǎng)基暗環(huán)境, 減緩培養(yǎng)基中IAA和IBA的降解[23]。

轉(zhuǎn)錄組測(cè)序是揭示某一物種特定組織或器官在某一狀態(tài)下的幾乎所有轉(zhuǎn)錄本序列信息的重要技術(shù)手段[24-26]。為揭示AC影響幼胚成苗的分子機(jī)制, 本研究利用轉(zhuǎn)錄組測(cè)序技術(shù)分析了在 MS[27]和 MSA (MS加 AC)培養(yǎng)基上離體培養(yǎng)的玉米幼胚苗地上部和地下部的基因表達(dá)情況, 并對(duì)部分表達(dá)差異顯著的基因進(jìn)行了實(shí)時(shí)熒光定量RT-PCR的驗(yàn)證, 旨在揭示AC促進(jìn)玉米幼胚發(fā)育成苗的基因表達(dá)機(jī)制, 為進(jìn)一步改進(jìn)玉米幼胚離體萌苗技術(shù)提供依據(jù)。

1 材料與方法

1.1 植物材料及處理

將玉米自交系昌 7-2種子播于溫室, 溫度控制在23～30℃, 自然光照。吐絲前套袋, 人工授粉14 d后, 取幼穗在無(wú)菌條件下剝?nèi)∮着? 盾片朝下接種于MS和MSA培養(yǎng)基上。

MSA培養(yǎng)基為添加AC 6 g L–1的MS培養(yǎng)基。MS和MSA兩種培養(yǎng)基均附加0.01 mg L–1NAA、0.04 mg L–16-BA、20 g L–1蔗糖和6 g L–1瓊脂。培養(yǎng)基分裝于100 mL三角瓶中, 每瓶約30 mL。每瓶接種6個(gè)幼胚, 每種培養(yǎng)基接種5瓶, 2個(gè)重復(fù)。接種后的幼胚培養(yǎng)于24℃、16 h光照/8 h黑暗的培養(yǎng)箱中。生長(zhǎng)9 d后, 從每瓶發(fā)育較為一致的幼苗中取2株, 調(diào)查幼苗根長(zhǎng)(最長(zhǎng)根的長(zhǎng)度)、根數(shù)、株高和單株鮮重, 對(duì)每種培養(yǎng)基處理調(diào)查10株, 2個(gè)重復(fù)。利用SPSS軟件對(duì)數(shù)據(jù)進(jìn)行兩個(gè)獨(dú)立樣本的 t檢驗(yàn),分析性狀的差異顯著性。從每種培養(yǎng)基上選取生長(zhǎng)一致的4株幼苗按地上部(莖和葉)和地下部(殘存盾片和根)取樣, 共4個(gè)樣品, 即MS-shoots、MS-roots、MSA-shoots和MSA-roots, 3個(gè)生物學(xué)重復(fù)。樣品經(jīng)液氮速凍后, -80℃保存, 用于RNA提取。

1.2 轉(zhuǎn)錄組測(cè)序

委托上海派森諾公司完成RNA提取、文庫(kù)構(gòu)建和轉(zhuǎn)錄組測(cè)序。利用TRIZOL試劑提取樣品總RNA,通過(guò)rRNA去除試劑盒純化得到mRNA, 構(gòu)建鏈特異性文庫(kù)。采用Illumina NextSeq 500平臺(tái)進(jìn)行雙末端測(cè)序, 得到原始序列(raw reads)。

1.3 基因表達(dá)數(shù)據(jù)分析

經(jīng)過(guò)去除接頭和低質(zhì)量reads的數(shù)據(jù)過(guò)濾, 得到Q ＞ 20的高質(zhì)量clean reads序列用于后續(xù)分析。參考基因組數(shù)據(jù)來(lái)源于 Ensembl數(shù)據(jù)庫(kù)(http://www. ensembl.org/)[28]。通過(guò)Bowtie2軟件建立參考基因組索引, 用Tophat2將clean reads比對(duì)到參考基因組上[29-30]。用HTSeq統(tǒng)計(jì)比對(duì)到每個(gè)基因上的Read Count值,作為基因的原始表達(dá)量[31]。用RPKM (Reads Per Kilo bases per Million reads)對(duì)表達(dá)量進(jìn)行標(biāo)準(zhǔn)化, 以RPKM值 ＞ 1為基因表達(dá)標(biāo)準(zhǔn)。用DESeq對(duì)基因表達(dá)進(jìn)行差異分析[32-33], 以表達(dá)倍數(shù)差異Fold change值 ＞ 2、顯著性P-value ＜ 0.05和FDR ＜ 0.05為差異基因篩選條件。用 GO功能富集分析對(duì)差異基因(differentially expressed genes, DEGs)進(jìn)行功能注釋,顯示DEGs顯著富集的功能分類[33]。用KEGG進(jìn)行生物通路分析確定 DEGs主要參與的代謝途徑和信號(hào)通路[35-36]。

1.4 熒光定量RT-PCR驗(yàn)證

通過(guò)分析轉(zhuǎn)錄組數(shù)據(jù), 選取 2種培養(yǎng)基上生長(zhǎng)的幼胚苗地下部的10個(gè)DEGs, 利用Primer 5.0軟件設(shè)計(jì)候選基因引物(表1)。以玉米甘油醛-3-磷酸脫氫酶(GAPDH)的編碼基因?yàn)閮?nèi)參, 進(jìn)行熒光定量RT-PCR驗(yàn)證。以RNA為模板, 用FastQuant cDNA第一鏈合成試劑盒(天根公司)合成 cDNA。用SuperReal熒光定量預(yù)混試劑(天根公司)配制反應(yīng)液,用7500 Real-time PCR System進(jìn)行熒光定量PCR,反應(yīng)程序?yàn)?5℃ 30 s; 95℃ 5 s, 60℃ 32 s, 40個(gè)循環(huán)。以 2–ΔΔCT方法計(jì)算基因的相對(duì)表達(dá)量。對(duì)每個(gè)基因的定量設(shè)置3個(gè)生物學(xué)重復(fù)。以MS-roots樣品中基因表達(dá)量為1, 統(tǒng)計(jì)MSA-roots中各基因的相對(duì)表達(dá)量, 用單樣本t檢驗(yàn)分析MSA-roots樣本中10個(gè)DEGs表達(dá)量是否達(dá)到顯著水平(P ＜ 0.05)。分析RNA-Seq和qRT-PCR兩平臺(tái)中10個(gè)DEGs相對(duì)表達(dá)量的相關(guān)性。

表1 用于qRT-PCR分析的10個(gè)基因及其引物Table 1 Primers used for qRT-PCR analysis of ten maize DEGs

2 結(jié)果與分析

2.1 AC顯著促進(jìn)玉米幼胚苗的生長(zhǎng)發(fā)育

胚齡14 d的玉米幼胚在MS和MSA培養(yǎng)基上培養(yǎng)9 d后, 幼苗長(zhǎng)勢(shì)差異明顯(圖1)。MSA培養(yǎng)基上的幼苗根長(zhǎng)、根數(shù)、株高和鮮重均顯著高于 MS培養(yǎng)基上的幼苗(P ＜ 0.05)(表 2)。在培養(yǎng)基中添加AC可以顯著促進(jìn)玉米幼胚離體培養(yǎng)成苗。

2.2 轉(zhuǎn)錄組數(shù)據(jù)分析

MS-roots、MS-shoots、MSA-roots和MSA-shoots分別有41 572 394、47 070 088、44 177 188和44 369 556個(gè)clean reads。共有130 209 524個(gè)clean reads比對(duì)到參考基因組上, 115 713 895個(gè)clean reads比對(duì)到基因區(qū)域, 其中96.96%比對(duì)到外顯子區(qū)域(表3)。

圖1 活性炭促進(jìn)玉米幼胚離體培養(yǎng)成苗Fig. 1 Active carbon accelerated the seedlings producing from immature embryos cultured in vitro

表2 MS和MSA培養(yǎng)基上玉米幼胚苗性狀的比較Table 2 Comparison of the traits of maize seedlings developing from immature embryos cultured on MS or MSA media

2.3 AC影響玉米幼胚苗地上部和地下部的基因表達(dá)

對(duì)MS和MSA培養(yǎng)基上玉米幼胚苗的基因表達(dá)進(jìn)行分析, 共發(fā)現(xiàn)2196個(gè)DEGs。其中, 地下部2142個(gè), 地上部147個(gè); 有93個(gè)基因在地上部和地下部中同時(shí)存在(圖2-A)。在2142個(gè)地下部DEGs中, 有1612個(gè)基因受AC上調(diào)表達(dá), 530個(gè)基因受AC下調(diào)表達(dá)。在147個(gè)地上部DEGs中, 有69個(gè)基因受AC上調(diào)表達(dá), 78個(gè)基因受AC下調(diào)表達(dá)(圖2-B)。這些結(jié)果表明, AC主要影響玉米胚苗地下部基因的表達(dá)。

表3 玉米幼胚苗的轉(zhuǎn)錄組數(shù)據(jù)分析Table 3 Analysis on transcriptome data of maize immature embryo seedlings

圖2 活性炭誘導(dǎo)的差異表達(dá)基因分布情況Fig. 2 Distribution of DEGs induced by active carbon

2.4 AC誘導(dǎo)的DEGs的GO富集分析

GO功能富集分析表明, 地下部的2142個(gè)DEGs中有 1630個(gè)基因獲得 GO注釋, 涉及生命進(jìn)程(biological process, BP)、細(xì)胞組分(cellular component, CC)和分子功能(molecular function, MF)三大類生物功能; 進(jìn)一步可細(xì)分成 131個(gè)顯著富集小類(terms)(P ＜ 0.05)。其中, DNA組裝(P = 4.36×10-19)、DNA組裝復(fù)合體(P = 7.75×10-18)和糖基水解酶活性(P = 9.02×10-13)分別是BP、CC和MF中最顯著富集的功能類別。地上部142個(gè)DEGs中有108個(gè)被注釋, 分別有16個(gè)BP、5個(gè)CC和15個(gè)MF Terms顯著富集(P ＜ 0.05), 最顯著富集的類別涉及脂類代謝過(guò)程(P = 4.25×10-6)、胞外區(qū)(P = 3.00×10-7)和過(guò)氧化物酶活性(P = 4.40×10-4)。地下部和地上部中DEGs涉及的20個(gè)最顯著富集的功能見(jiàn)圖3。

2.5 AC誘導(dǎo)的DEGs的KEGG富集分析

KEGG富集分析表明, 地下部DEGs注釋到108個(gè)代謝通路。其中, 56個(gè)涉及能量、碳水化合物、氨基酸、脂類和次生代謝物等代謝途徑; 11個(gè)涉及細(xì)胞周期、p53信號(hào)通路、肌動(dòng)蛋白細(xì)胞骨架調(diào)節(jié)等細(xì)胞進(jìn)程途徑; 13個(gè)涉及植物激素信號(hào)轉(zhuǎn)導(dǎo)、MAPK信號(hào)通路和 AMPK信號(hào)通路等環(huán)境信息進(jìn)程途徑; 8個(gè)涉及DNA復(fù)制、堿基切除修復(fù)和核糖體等遺傳信息進(jìn)程途徑; 20個(gè)涉及黃體酮調(diào)節(jié)卵母細(xì)胞成熟、植物-病原菌互作、趨化因子信號(hào)通路等有機(jī)系統(tǒng)途徑。20個(gè)最顯著富集的代謝通路主要是光合作用-天線蛋白、苯丙烷類物質(zhì)生物合成和細(xì)胞周期途徑。地上部DEGs涉及10個(gè)代謝通路, 最顯著富集途徑為泛醌和其他萜-醌類物質(zhì)合成(圖4)。

圖3 活性炭誘導(dǎo)的玉米胚苗地上部和地下部DEGs中各涉及的20個(gè)最顯著富集GO分類Fig. 3 Twenty most significantly enriched Gene Ontology (GO) terms of the DEGs induced by active carbon in roots and shoots of maize seedlings

對(duì)地下部富集的細(xì)胞周期途徑、植物激素轉(zhuǎn)導(dǎo)途徑和外源物質(zhì)代謝途徑中的DEGs進(jìn)一步分析。細(xì)胞周期途徑涉及 7個(gè) DEGs上調(diào)表達(dá), 分別為細(xì)胞周期蛋白 A2、細(xì)胞周期蛋白 B2、細(xì)胞周期超家族蛋白、細(xì)胞分裂周期蛋白 CDH1、增殖細(xì)胞核抗原PCNA、微小染色體維持缺陷蛋白MCM3和有絲分裂檢紡錘體監(jiān)測(cè)點(diǎn)蛋白 BUBR1的編碼基因。在植物激素轉(zhuǎn)導(dǎo)途徑中, 生長(zhǎng)素信號(hào)轉(zhuǎn)導(dǎo)中的 2個(gè)Aux/IAA編碼基因上調(diào)表達(dá), 水楊酸信號(hào)轉(zhuǎn)導(dǎo)中的具有bZIP結(jié)構(gòu)的TGA轉(zhuǎn)錄因子基因下調(diào)表達(dá)。在外源物質(zhì)代謝途徑中, 細(xì)胞色素 P450、乙醛脫氫酶3B1、谷胱氨肽轉(zhuǎn)移酶7和谷胱氨肽轉(zhuǎn)移酶27的編碼基因表達(dá)上調(diào), 乙醇脫氫酶2基因表達(dá)下調(diào)(表4)。

圖4 地下部和地上部樣品中DEGs富集的KEGG途徑Fig. 4 Enriched KEGG pathways of DEGs in the roots and shoots samples

2.6 DEGs的實(shí)時(shí)熒光定量RT-PCR驗(yàn)證

對(duì) 2種培養(yǎng)基上生長(zhǎng)的幼胚苗地下部中 10個(gè)DEGs進(jìn)行了熒光定量 RT-PCR驗(yàn)證。以 MS-roots樣品中基因表達(dá)量為1, 統(tǒng)計(jì)MSA-roots中各基因的相對(duì)表達(dá)量。結(jié)果表明, qRT-PCR中的基因表達(dá)變化與 RNA-seq結(jié)果一致(圖 5)。經(jīng)單樣本 t檢驗(yàn), MSA-roots樣品中10個(gè)DEGs表達(dá)量與MS樣品中表達(dá)量均差異顯著。將RNA-seq和qRT-PCR中基因差異表達(dá)量進(jìn)行 log2轉(zhuǎn)化后進(jìn)行相關(guān)性分析, 皮爾森相關(guān)系數(shù)為0.922, 在0.01水平上顯著相關(guān)(圖6),說(shuō)明RNA-seq的結(jié)果可靠。

經(jīng) qRT-PCR驗(yàn)證, 組蛋白 H4 (GRMZM2

G149178)、細(xì)胞周期蛋白 CYCB2 (GRMZM2G 138886)、細(xì)胞周期蛋白激酶 CDK (GRMZM2G 068193)、增殖細(xì)胞核抗原 PCNA2 (GRMZM2G 108712)、木質(zhì)素過(guò)氧化物酶 POX72 (GRMZM2G 089982)、GRAS轉(zhuǎn)錄因子 GRAS19 (GRMZM2G1 72657)和富亮氨酸重復(fù)類受體蛋白激酶 LRR-RLK (GRMZM2G048294)受 AC影響在地下部中上調(diào)表達(dá)。MAP激酶(GRMZM2G017792)、木質(zhì)素過(guò)氧化物酶 POX2 (GRMZM2G089982)和 β-糖苷酶(GRM ZM2G055699)受AC影響在地下部中下調(diào)表達(dá)。

表4 細(xì)胞周期、植物激素轉(zhuǎn)導(dǎo)和外源物質(zhì)降解代謝途徑中的DEGsTable 4 DEGs functioning in cell cycle, plant hormone signal transduction, xenobiotics bio-degradation and metabolism pathways

圖5 受活性炭誘導(dǎo)的10個(gè)DEGs的qRT-PCR檢測(cè)Fig. 5 qRT-PCR verification of ten DEGs induced by active carbon

圖6 10 DEGs的RNA-seq與qRT-PCR結(jié)果相關(guān)性分析Fig. 6 Correlation analysis of the expression changes of ten DEGs as revealed by RNA-seq and qRT-PCR, respectively

3 討論

本研究利用Illumina 500測(cè)序技術(shù)對(duì)AC促進(jìn)玉米幼胚發(fā)育成苗的基因表達(dá)情況進(jìn)行了轉(zhuǎn)錄組分析。測(cè)序數(shù)據(jù)質(zhì)量檢測(cè)表明, 高質(zhì)量數(shù)據(jù)(Q20)在97.34%以上, 可以保證后續(xù)分析結(jié)果的準(zhǔn)確性。選取的10個(gè)DEGs的qRT-PCR結(jié)果與轉(zhuǎn)錄組分析一致, 表明轉(zhuǎn)錄組測(cè)序結(jié)果可信度高。

AC促進(jìn)玉米胚苗的生長(zhǎng)發(fā)育可能與控制細(xì)胞分裂的基因調(diào)控表達(dá)上調(diào)有密切關(guān)系。本研究中, AC除了影響地下部細(xì)胞周期途徑中的CYC、CDH、PCNA、MCM、BUB基因上調(diào)表達(dá)外, 還影響其他與細(xì)胞增殖相關(guān)基因顯著上調(diào)表達(dá), 如H4、CDK、GRAS等。前人研究表明, 組蛋白是染色體的組成部分, 其翻譯后修飾可引起染色質(zhì)結(jié)構(gòu)重塑, 在基因表達(dá)調(diào)控中起重要作用[37-38]。CYC和 CDK通過(guò)特異性結(jié)合調(diào)控細(xì)胞周期, 控制細(xì)胞增殖, 是細(xì)胞周期調(diào)控的主要因子[39]。PCNA是DNA多聚酶的一種輔助蛋白質(zhì), 在DNA復(fù)制、DNA修復(fù)和細(xì)胞周期中發(fā)揮重要作用, 是細(xì)胞增殖活性的重要指標(biāo)[40-42]。 MCM是與DNA復(fù)制和細(xì)胞增殖密切相關(guān)的蛋白家族, 在真核細(xì)胞的DNA復(fù)制中, 形成復(fù)制前復(fù)合物,具有DNA解鏈酶活性, 啟動(dòng)DNA復(fù)制, 是調(diào)控細(xì)胞由G1期進(jìn)入S期的關(guān)鍵蛋白, 是反映細(xì)胞增殖活性的特異性指標(biāo)[43-44]。GRAS轉(zhuǎn)錄因子是植物特有的轉(zhuǎn)錄因子, 在植物生長(zhǎng)發(fā)育、信號(hào)轉(zhuǎn)導(dǎo)、解毒、生物和非生物脅迫相關(guān)應(yīng)答過(guò)程中起作用[45-46]。上述結(jié)果表明, 在培養(yǎng)基中添加 AC可以調(diào)控細(xì)胞增殖相關(guān)的基因表達(dá), 從而促進(jìn)玉米幼胚苗的生長(zhǎng)與發(fā)育。

激素對(duì)植物發(fā)育具有重要的調(diào)節(jié)作用。本研究中AC促進(jìn)幼胚苗根部生長(zhǎng)素(Auxin)信號(hào)轉(zhuǎn)導(dǎo)途徑中Aux/IAA基因上調(diào)表達(dá)。Auxin影響胚發(fā)生、根發(fā)育、葉片伸展和果實(shí)成熟等許多生長(zhǎng)發(fā)育過(guò)程[47-48]。Aux/IAA蛋白在Auxin信號(hào)轉(zhuǎn)導(dǎo)途徑中起主要的調(diào)控作用, Auxin濃度低時(shí), Aux/IAA蛋白與生長(zhǎng)素反應(yīng)因子ARF結(jié)合, 抑制下游基因的表達(dá), Auxin濃度高時(shí), Aux/IAA蛋白泛素化降解, ARF激活下游基因表達(dá)[49]。本研究中AC促進(jìn)水楊酸(salicylic acid, SA)信號(hào)轉(zhuǎn)導(dǎo)途徑中 TGA轉(zhuǎn)錄因子基因下調(diào)表達(dá)。SA作為植物內(nèi)源信號(hào)分子, 主要參與應(yīng)對(duì)生物和非生物脅迫過(guò)程, 提高植物抵抗能力[50]。TGA是SA信號(hào)轉(zhuǎn)導(dǎo)途徑中的重要轉(zhuǎn)錄因子, 與病程相關(guān)蛋白基因非表達(dá)子 1 (nonexpressor of pathogenesis-related protein gene 1, NPR1)相互作用, 激活病程相關(guān)蛋白1 (pathogenesis-related protein 1, PR1)表達(dá), 最終誘導(dǎo)系統(tǒng)獲得性抗性(systemic acquired resistance, SAR)的產(chǎn)生[51-52]。AC 可以促進(jìn)胚苗地下部中CYP450編碼基因的上調(diào)表達(dá)。CYP450是一種末端加氧酶, 作為植物中最大的酶家族廣泛參與植物體內(nèi)多種物質(zhì)合成和降解過(guò)程, 在催化外源物質(zhì)降解過(guò)程中具有重要作用[53]。

4 結(jié)論

發(fā)現(xiàn) AC能顯著促進(jìn)玉米幼胚離體培養(yǎng)幼苗的生長(zhǎng)和發(fā)育, 并從轉(zhuǎn)錄組水平上揭示了 AC促進(jìn)玉米幼胚離體成苗的基因表達(dá)情況。AC可引起玉米幼胚苗中2196個(gè)基因的差異表達(dá), 包括地下部的2142個(gè)基因和地上部的147個(gè)基因。地下部中DEGs主要涉及光合作用-天線蛋白、細(xì)胞循環(huán)、植物激素信號(hào)轉(zhuǎn)導(dǎo)、外源物質(zhì)代謝等108條KEGG途徑; 地上部DEGs涉及10條KEGG途徑, 最顯著富集的為泛醌和其他萜-醌類物質(zhì)合成途徑。上述結(jié)果對(duì)揭示玉米幼胚萌發(fā)的分子機(jī)理和進(jìn)一步改進(jìn)玉米幼胚離體培養(yǎng)條件提供了依據(jù)。

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Transcriptome Analysis of Promotive Effects of Active Carbon on Growth and Development of Maize Seedlings from in vitro Cultured Immature Embryos

WANG Jin-Ping1,2, SUN Guo-Zhong2,*, and WANG Hai-Bo2,*
1College of Agronomy, Agricultural University of Hebei, Baoding 071001, China;2Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences / Plant Genetic Engineering Center of Hebei Province, Shijiazhuang 050051, China

Immature embryos from the maize inbred line Chang 7-2 were collected at 14 days after pollination, and cultured on MS or MSA medium (MS medium plus active carbon) for nine days at 24°C. Active carbon significantly accelerated the growth and development of maize seedlings from cultured immature embryos. Using RNA-seq technique, the genes involved in the growth promotive effects of active carbon were analyzed. The presence of active carbon in the medium affected the gene expression in seedlings significantly. Number of up- and down-regulated genes was 1612 and 530 in roots, as well as 69 and 78 in shoots, respectively; indicating that active carbon mainly affects gene expression in roots. GO enrichment analysis showed that differentially expressed genes (DEGs) in roots were mainly involved in DNA packaging, DNA packaging complex and hydrolase activity; the DEGs in shoots were mainly involved in lipid metabolic process, extracellular region and peroxidase activity. The KEGG enrichment analysis showed that the DEGs in roots were significantly associated with energy metabolism, carbohydrate metabolism, lipid metabolism, amino acid metabolism, cell cycle and plant hormone signal transduction. The DEGs in shoots were significantly associated with biosynthesis of ubiquinone and other terpenoid-quinone compounds. Several key genes involved in the cell cycle pathway (i.e., CYC, CDH1, MCM3, PCNA2, and BUB1), signal transduction of auxin (Aux/IAA) and cytochrome function (CYP450 oxidase) were significantly up-regulated by active carbon. Ten DEGs were confirmed by Real-time quantitative PCR assay, suggesting that our data and analysis of transcriptome sequencing are reliable.

Maize; Immature embryo; Active carbon; Transcriptome

(

): 2017-03-21; Accepted(接受日期): 2017-04-20; Published online(網(wǎng)絡(luò)出版日期): 2017-05-08.

10.3724/SP.J.1006.2017.01489

本研究由河北省財(cái)政專項(xiàng)(2009055001, F15R25)資助。

This study was supported by the Financial Fund Program of Hebei Province (2009055001, F15R25).

*通訊作者(Corresponding authors): 孫果忠, E-mail: 13933023804@163.com; 王海波, E-mail: nkywanghb@163.com第一作者聯(lián)系方式: E-mail: 15226506557@163.com

URL: http://kns.cnki.net/kcms/detail/11.1809.S.20170508.1007.012.html