王菲菲 張勝忠 胡曉輝 CHU Ye 崔鳳高 鐘 文 趙立波 張?zhí)煊?郭進(jìn)濤 于豪諒 苗華榮,* 陳 靜,*

比較轉(zhuǎn)錄組分析花生種子休眠調(diào)控網(wǎng)絡(luò)

王菲菲1張勝忠1胡曉輝1CHU Ye2崔鳳高1鐘 文3趙立波4張?zhí)煊?郭進(jìn)濤5于豪諒6苗華榮1,*陳 靜1,*

1山東省花生研究所, 中國(guó)山東青島 266100;2佐治亞大學(xué)蒂芙頓校區(qū)園藝系, 美國(guó)佐治亞州蒂芙頓 31793;3山東省種子管理總站, 中國(guó)山東濟(jì)南 250100;4青島市農(nóng)業(yè)技術(shù)推廣中心, 中國(guó)山東青島 266071;5新鄭市和莊農(nóng)業(yè)服務(wù)中心, 中國(guó)河南新鄭 451150;6煙臺(tái)楓林食品股份有限公司, 中國(guó)山東煙臺(tái) 264108

種子休眠性是花生重要且復(fù)雜的農(nóng)藝性狀, 對(duì)花生(L.)的產(chǎn)量和品質(zhì)影響巨大。為深入揭示花生種子休眠維持和解除的分子調(diào)控網(wǎng)絡(luò), 本研究以強(qiáng)休眠品種花育52號(hào)(HY52)和弱休眠突變株系M23、M67為試材, 種子吸脹處理(0 h、12 h、24 h)后測(cè)定其激素ABA和GA含量并進(jìn)行轉(zhuǎn)錄組測(cè)序。吸脹12 h時(shí)M23和M67中GA含量顯著高于HY52, ABA含量和ABA/GA比值則低于HY52。測(cè)序共得到31,373個(gè)差異表達(dá)基因(DEGs), 其中ABA和GA生物合成和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的基因在種子休眠維持和解除過程中發(fā)生顯著變化, 挖掘到50個(gè)ABA相關(guān)基因、8個(gè)GA相關(guān)基因、49個(gè)乙烯相關(guān)基因和13個(gè)生長(zhǎng)素相關(guān)基因。此外, 還鑒定到許多參與碳水化合物和脂質(zhì)代謝、氨基酸代謝途徑相關(guān)的DEG, 挖掘到糖代謝相關(guān)基因5個(gè)、脂質(zhì)代謝相關(guān)基因4個(gè); 晝夜節(jié)律調(diào)控也可能參與花生種子休眠解除。這表明, 花生種子休眠維持和解除的調(diào)控是一個(gè)復(fù)雜網(wǎng)絡(luò), 植物激素平衡調(diào)控可能只是其中一個(gè)重要部分。

花生; 休眠維持; 休眠解除; 轉(zhuǎn)錄組; 植物激素; 氨基酸代謝

種子休眠是指在特定時(shí)間范圍內(nèi)、適宜的生長(zhǎng)環(huán)境下, 具備萌發(fā)能力的種子不發(fā)芽的現(xiàn)象, 是植物為抵抗和適應(yīng)不良環(huán)境的一種適應(yīng)性性狀[1-2]。它是植物長(zhǎng)期進(jìn)化過程中形成的自我保護(hù)機(jī)制, 是植物界中普遍存在的特性[3]。種子休眠是極其重要和復(fù)雜的農(nóng)藝性狀, 對(duì)農(nóng)業(yè)安全生產(chǎn)有重要意義。農(nóng)作物如小麥、大麥、水稻和花生等在長(zhǎng)期馴化、培育過程中, 注重追求高產(chǎn)高效而忽視了保留適度的種子休眠性, 導(dǎo)致種子休眠性減弱甚至喪失, 使得產(chǎn)量和食用品質(zhì)下降、種用安全隱患(易受黃曲霉侵染, 產(chǎn)生黃曲霉毒素)等一系列問題[4-5]。因此, 揭示種子休眠調(diào)控機(jī)制并選育具有適度休眠性的農(nóng)作物品種/系是作物育種家重要的研究方向之一。

目前, 關(guān)于種子休眠的生物學(xué)機(jī)制仍未得到全面解析, 種子會(huì)隨著儲(chǔ)藏時(shí)間的增加而逐漸失去休眠性[6-7]。早在50多年前就已報(bào)道, 植物激素脫落酸(abscisic acid, ABA)和赤霉素(gibberellin, GA)及其兩者間的平衡在調(diào)控種子休眠和萌發(fā)中發(fā)揮主要作用[8-10]。ABA誘導(dǎo)和維持種子休眠而GA則促進(jìn)休眠解除和種子萌發(fā)[7], 在種子休眠和萌發(fā)過程中, 一系列ABA和GA合成、代謝及信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的基因表達(dá)發(fā)生變化[7]。除此之外, 其他植物激素如乙烯(ethylene, ETH)、生長(zhǎng)素(auxin)、細(xì)胞分裂素(cytokinins, CTK)和油菜素內(nèi)酯(brassinosteroids, BR)也在調(diào)控種子休眠和萌發(fā)中發(fā)揮重要作用[7,11]。種子萌發(fā)和休眠受多個(gè)因子調(diào)控, 包括植物激素和休眠蛋白等, 這些調(diào)控信息將在種子吸脹過程中被整合處理, 最終決定是否萌發(fā)[10]。

花生是食用油和植物蛋白的重要來源, 是我國(guó)重要的油料和經(jīng)濟(jì)作物。由于花生地上開花地下結(jié)果, 其種子休眠調(diào)控更加復(fù)雜, 為花生種子休眠研究帶來更多挑戰(zhàn)。前人研究主要集中在花生休眠品種篩選鑒定、相關(guān)QTL定位[4,12-14], 但是花生休眠關(guān)鍵基因的挖掘及克隆相對(duì)滯后。轉(zhuǎn)錄組測(cè)序是高通量篩選差異表達(dá)基因的有效方法, 利用轉(zhuǎn)錄組分析種子休眠和萌發(fā)的研究已有報(bào)道[15-18], Xu等[19]對(duì)休眠不深的魯花14號(hào)的新收獲種子(freshly harvested seed, FS)、后熟種子(after-ripening seed, DS)和新萌發(fā)種子(newly germinatedseed, GS)進(jìn)行比較轉(zhuǎn)錄組測(cè)序分析, 在GS vs DS的比較組中, 許多上調(diào)基因與植物激素合成和信號(hào)轉(zhuǎn)導(dǎo)相關(guān), 包括生長(zhǎng)素信號(hào)、油菜素內(nèi)酯合成和信號(hào)轉(zhuǎn)導(dǎo)及GA、ABA信號(hào)轉(zhuǎn)導(dǎo)的大部分元件[19]。通過測(cè)定下胚軸和胚根、胚芽、子葉中的激素含量, 發(fā)現(xiàn)在種子吸脹過程中, ABA含量逐步下降, ABA/GA3比值在吸脹4 h達(dá)到最大值, 隨后下降。其他物種也有相關(guān)報(bào)道, 低溫處理能打破豆梨(Dence)休眠, 對(duì)低溫處理的種子進(jìn)行轉(zhuǎn)錄組測(cè)序分析發(fā)現(xiàn), 多個(gè)差異表達(dá)基因(differentially expressed genes, DEGs)與植物激素(ABA, GA, CTK, ETH, BR, JA)的代謝和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)。在種子休眠解除過程中, 與淀粉和蔗糖代謝、脂質(zhì)代謝相關(guān)的基因以及編碼轉(zhuǎn)錄因子的基因也有差異表達(dá)[20]。

盡管種子休眠維持和解除的基因調(diào)控網(wǎng)絡(luò)及在擬南芥和谷類作物中取得了巨大突破[20-21], 但有關(guān)雙子葉植物花生種子休眠和萌發(fā)的機(jī)制尚不清楚。為揭示花生種子休眠維持和解除機(jī)制, 本研究選用強(qiáng)休眠品種花育52號(hào)(Huayu 52, HY52)和由HY52誘變而來的2個(gè)弱休眠材料M23和M67為試材, 吸脹處理(0 h、12 h、24 h)后進(jìn)行轉(zhuǎn)錄組測(cè)序。同時(shí)對(duì)HY52進(jìn)行全長(zhǎng)轉(zhuǎn)錄組測(cè)序, 以獲得更好的基因功能注釋。同時(shí)測(cè)定了種子中ABA和GA含量, 分析種子吸脹過程中植物激素變化, 旨在揭示花生休眠維持和解除過程中關(guān)鍵基因的完整表達(dá)譜, 為花生適度休眠育種提供新的思路。

1 材料與方法

1.1 植物材料及生長(zhǎng)條件

試驗(yàn)材料系花育52號(hào)(Huayu 52, HY52)及經(jīng)EMS誘變而來的M4突變體M23和M67[5], 其中花育52號(hào)為山東省花生研究所育成的花生品種, 具有強(qiáng)休眠性, M23和M67則為2個(gè)具有弱休眠性的突變材料。試驗(yàn)材料種植在山東省萊西市實(shí)驗(yàn)農(nóng)場(chǎng)(36.86°N, 120.53°E), 種植方式為起壟雙行單粒播種,行距40.0 cm, 株距16.7 cm, 覆膜。5月上旬播種, 種子成熟后收獲晾曬10 d后進(jìn)行種子休眠性檢測(cè), 剩余種子置于紙袋中室溫保存。參照胡曉輝等[5]的方法, 以種子萌發(fā)率來評(píng)價(jià)種子休眠性強(qiáng)弱。

1.2 cDNA文庫構(gòu)建、測(cè)序和數(shù)據(jù)分析

本研究選擇對(duì)種子進(jìn)行吸脹處理, 分析該時(shí)期種子激素含量及轉(zhuǎn)錄組信息變化?；诤鷷暂x等[5]前期研究, 選擇吸脹0 h、12 h和24 h 3個(gè)處理進(jìn)行深入分析。每個(gè)供試材料選取飽滿無損的種子10粒, 置于培養(yǎng)皿中, 吸脹0 h、12 h、24 h時(shí)分別取樣用于RNA提取并構(gòu)建轉(zhuǎn)錄組文庫, 轉(zhuǎn)錄組測(cè)序設(shè)3個(gè)生物學(xué)重復(fù)。以TRIzol法進(jìn)行RNA提取并進(jìn)行DNase I處理, 質(zhì)檢合格后送杭州聯(lián)川生物技術(shù)股份有限公司建庫測(cè)序。二代轉(zhuǎn)錄組測(cè)序平臺(tái)為Illumina HiSeq4000, HY52的全長(zhǎng)轉(zhuǎn)錄組測(cè)序平臺(tái)為PacBio Sequel。

1.3 差異表達(dá)基因和富集分析

測(cè)序產(chǎn)生的原始數(shù)據(jù)(rawdata)經(jīng)cutadapt過濾得到有效數(shù)據(jù)(cleandata)。所有有效數(shù)據(jù)經(jīng)Hisat v2.0比對(duì)到栽培花生參考基因組上(https://peanutbase. org/peanut_genome)[22]。計(jì)算每個(gè)轉(zhuǎn)錄本的FPKM(每千堿基轉(zhuǎn)錄序列的預(yù)期片段數(shù)/百萬堿基對(duì)測(cè)序)值來表示歸一化的基因表達(dá)量。根據(jù)公式(表達(dá)倍數(shù)=FPKMA/FPKMB)計(jì)算基因表達(dá)量的倍數(shù)變化, 將表達(dá)差異倍數(shù)log2|Fold Change|>1且FDR<0.05的基因定義為差異表達(dá)基因(DEGs)。差異表達(dá)基因基于GO (Gene Ontology)數(shù)據(jù)庫和KEGG (Kyoto Encyclopedia of Genes and Genomes)數(shù)據(jù)庫進(jìn)行功能注釋并按照生物過程(biological process, BP)、細(xì)胞組分(cellular component, CC)和分子功能(molecular function, MF)對(duì)其富集分析, 找出差異基因顯著富集的GO term和KEGG term?；诙虝r(shí)間序列表達(dá)式挖掘(short time-series expression minor, STME)聚類方法將差異基因聚類到不同的表達(dá)譜中[23]。

1.4 數(shù)據(jù)處理及作圖

用Graphpad Prism 8軟件對(duì)測(cè)定數(shù)據(jù)進(jìn)行顯著性檢驗(yàn)和繪圖。使用聯(lián)川生物云平臺(tái)在線工具(https://www.omicstudio.cn/tool)進(jìn)行差異基因GO功能分析和KEGG分析。

2 結(jié)果與分析

2.1 HY52與M23、M67的休眠表型差異

HY52萌發(fā)率為35%, 而M23和M67萌發(fā)率分別為78.33%和83.33%, 三者萌發(fā)率間有顯著差異, HY52為強(qiáng)休眠品種, M23和M67為弱休眠材料(圖1-A)。3種材料中GA含量均隨吸脹時(shí)間增加而升高(圖1-B), 而ABA含量則呈下降趨勢(shì)(圖1-C)。吸脹0 h時(shí), GA和ABA含量在HY52、M23、M67之間并無顯著差異, 而吸脹12 h和24 h時(shí)M23和M67中GA含量顯著高于HY52 (圖1-B), M67中ABA含量和ABA/GA比值則顯著低于HY52 (圖1-C, D), 吸脹12 h時(shí)M23中ABA含量和ABA/GA比值也低于HY52 (圖1-C, D)。這與HY52、M23、M67的休眠表型一致。種子吸脹過程中, HY52、M23、M67中ABA/GA比值分別從1335、986、676降至676、651、343 (圖1-D)。

圖1 HY52、M23和M67的休眠性差異

A: HY52、M23和M67的萌發(fā)率, B～D: 吸脹12 h和24 h的GA含量、ABA含量和ABA/GA。GA: 赤霉素; ABA: 脫落酸; HY52: 花育52號(hào)。星號(hào)表示通過單因素方差分析與HY52相比有顯著性(*<0.05, **<0.01, ***<0.001)。

A: germination rate of dormant peanut variety HY52, weak dormant accessions M23 and M67, B–D: GA content, ABA content and ABA/GA of HY52, M23, and M67 after imbibition at 0, 12, and 24 h, respectively. GA: gibberellin; ABA: abscisic acid; HY52: Huayu 52. Asterisks indicate significant differences compared to HY52 by one-way ANOVA (*<0.05, **<0.01, and ***<0.001).

2.2 轉(zhuǎn)錄組測(cè)序整體分析

本研究供試的27個(gè)樣本, 共獲得原始序列數(shù)目大約為39,391,916～63,130,436。經(jīng)過濾, 有效序列數(shù)目為39,016,270～62,578,018, 有效堿基為5.98～9.39 Gb。平均GC含量為46.38%, 有效序列的Q30超過94.81%。有效序列的比率為88.92%～99.20%, 比對(duì)到基因組上的平均比率超過92.51% (表1)。另外, 為了對(duì)差異表達(dá)基因進(jìn)行更好的注釋, 對(duì)休眠品種HY52進(jìn)行了全長(zhǎng)轉(zhuǎn)錄組測(cè)序, 在KOG (Kyoto Encyclopedia of Genes and Genomes)、KEGG (Kyoto Encyclopedia of Genes and Genomes)、NR (Non- redundant Protein Database)、Swiss Prot (Non- redundant Protein Database)和GO (Gene Ontology)數(shù)據(jù)庫中都進(jìn)行了功能注釋(表2)。

表1 參試樣品的轉(zhuǎn)錄組數(shù)據(jù)質(zhì)量

表2 花育52號(hào)全長(zhǎng)轉(zhuǎn)錄組的功能注釋

2.3 轉(zhuǎn)錄組差異表達(dá)基因及表達(dá)譜分析

根據(jù)<0.05和|log2FC|≥1的標(biāo)準(zhǔn), 所有樣本中共鑒定到15,746個(gè)差異表達(dá)基因。HY52吸脹12 h后有1392個(gè)上調(diào)基因和1702個(gè)下調(diào)基因, 吸脹24 h后有2001 (上調(diào))/2075 (下調(diào))個(gè)差異基因。M23吸脹12 h和24 h的差異表達(dá)基因數(shù)分別為1766/1522和1384/2498, M67吸脹12h和24 h的差異表達(dá)基因數(shù)分別為1868/2796和2061/2368 (圖2-A)。韋恩圖展示了吸脹和對(duì)照組中相同和特異的差異表達(dá)基因數(shù), HY52和M23的4個(gè)比較組中共有的上調(diào)基因有40個(gè)(圖2-C), 下調(diào)基因有15個(gè)(圖2-D); HY52和M67的4個(gè)比較組中共有的上調(diào)基因有61個(gè)(圖2-E), 下調(diào)基因有51個(gè)(圖2-F)。3個(gè)材料的比較組中, M23 (M23_12 vs M23_0, M23_24 vs M23_0)和M67 (M67_12 vs M67_0, M67_24 vs M67_0)中的特有差異表達(dá)基因數(shù)量比HY52 (HY52_12 vs HY52_0, HY52_24 vs HY52_0)中多(圖2-C～F), 說明M23和M67在吸脹過程中有更多的基因參與休眠解除。另外, 在吸脹12 h時(shí)HY52和M23中上調(diào)的差異表達(dá)基因數(shù)是156, 下調(diào)的差異表達(dá)基因數(shù)是116, 吸脹24 h時(shí)分別為287 (上調(diào))和532 (下調(diào)); HY52和M67在吸脹12 h和24 h的差異表達(dá)基因數(shù)分別為272 (上調(diào))/335 (下調(diào))和542 (上調(diào))/459 (下調(diào))。

為深入探究在種子休眠解除過程中的基因表達(dá)譜變化, 利用STEM算法[23]將HY52中52,747個(gè)差異表達(dá)基因、M23中的54,233個(gè)差異表達(dá)基因和M67中的53,060個(gè)差異表達(dá)基因聚類到16個(gè)表達(dá)譜中。根據(jù)顯著性分析將HY52中26,816個(gè)差異表達(dá)基因聚類到6個(gè)表達(dá)譜中: 5個(gè)上調(diào)的表達(dá)譜(profiles 11, 15, 13, 12, 8)和一個(gè)雙相表達(dá)譜(profile 14) (圖3-A)。與之類似, M23中25,999個(gè)差異表達(dá)基因聚類到7個(gè)表達(dá)譜中, M67中24,681個(gè)差異表達(dá)基因聚類到6個(gè)表達(dá)譜中(圖3-B, C)。結(jié)果表明, 3個(gè)材料中有5個(gè)相同的上調(diào)表達(dá)譜, 包括11、15、13、12和14, 而2個(gè)下調(diào)表達(dá)譜4和0只出現(xiàn)在M23中, 表達(dá)譜8則在HY52和M67中同時(shí)存在。M23中上調(diào)基因數(shù)目(profiles 11和15)大于HY52中上調(diào)基因數(shù)量, M67中上調(diào)基因數(shù)目(profiles 15, 13, 12和8)大于HY52和M23在這幾個(gè)表達(dá)譜中的基因數(shù)量。M23和M67都是弱休眠材料卻有不同表達(dá)譜, 可能是由突變位點(diǎn)的差異導(dǎo)致。

圖2 不同處理差異表達(dá)基因比較及韋恩圖

HY52: 花育52號(hào); DEGs: 差異表達(dá)基因。HY52: Huayu 52; DEGs: differentially expressed genes.

圖3 HY52、M23和M67的差異基因表達(dá)譜

HY52 (A)和M67 (C)的表達(dá)譜聚成2個(gè)組, 即上調(diào)和雙向表達(dá)譜, M23 (B)則聚成3個(gè)組, 即上調(diào)、下調(diào)和雙向表達(dá)譜。表達(dá)譜編號(hào)標(biāo)記在左上角, 每個(gè)表達(dá)譜對(duì)應(yīng)的值標(biāo)記在左下角, 括號(hào)中為每個(gè)表達(dá)譜中差異基因的數(shù)量。HY52: 花育52號(hào)。

Profiles of HY52 (A) and M67 (C) were clustered into two groups, namely Up (upregulated) and Bi (biphasic expression pattern), however, M23 (B) were clustered into three groups, namely Up (upregulated), Down (downregulated), and Bi (biphasic expression pattern). Profile numbers are indicated in the top left-hand corner, and the corresponding-values for each profile are shown in the bottom left-hand corner. The number of DEGs with each profile is shown in the brackets. HY52: Huayu 52.

2.4 花生休眠維持相關(guān)差異表達(dá)基因的富集分析

為揭示種子休眠維持的機(jī)制, 對(duì)6個(gè)比較組(HY52_0 vs M23_0, HY52_0 vs M67_0, HY52_12 vsM23_12, HY52_24 vs M23_24, HY52_12 vs M67_12, HY52_24 vs M67_24)進(jìn)行了GO富集分析。與預(yù)期一致, 結(jié)果表明激素和脅迫相關(guān)的GO條目在HY52 vs M23和HY52 vs M67的比較組中顯著富集, 如脫落酸刺激的細(xì)胞反應(yīng)(cellular response to abscisic acid stimulus)、赤霉素反應(yīng)(response to gibberellin)、生長(zhǎng)素反應(yīng)(response to auxin)、脅迫反應(yīng)(response to stress)、鹽脅迫反應(yīng)(response to salt stress)、脫落酸激活的信號(hào)通路(abscisic acid- activated signaling pathway)、茉莉酸介導(dǎo)的信號(hào)通路(jasmonic acid mediated signaling pathway)、乙烯結(jié)合(ethylene binding)、乙烯激活的信號(hào)通路(ethylene- activated signaling pathway) (圖4)。除此之外, 還有很多防御相關(guān)的條目, 如防御反應(yīng)(defense response)、活性氧代謝的正調(diào)控(positive regulation of reactive oxygen species metabolic process)、種子耐干燥性的獲得(acquisition of desiccation tolerance in seed)。氨基酸代謝相關(guān)的條目包括硫胺素生物合成(thiamine biosynthetic process)、谷氨酸代謝過程(glutamate metabolic process)和天冬氨酸代謝過程(aspartate metabolic process)。碳水化合物代謝相關(guān)的條目也參與這個(gè)過程, 如半乳糖代謝過程(galactose metabolic process)、阿拉伯糖合成過程(arabinose biosynthetic process)、半乳糖氨基轉(zhuǎn)移酶活性(galactinol-sucrose galacosyl-transfererase activity)。另外還富集到晝夜節(jié)律的負(fù)調(diào)控(negative regulation of circadian rhythm)、脂肪酸合成過程(fatty acid biosynthetic process)和三羧酸循環(huán)酶復(fù)合體(tricarboxylicacid cycle enzyme complex)等條目。

圖4 花生種子休眠保持過程中差異基因GO富集分析

Fig. 4 GO enrichment of the DEGs during peanut dormancy maintenance period

HY52: 花育52號(hào)。HY52: Huayu 52.

為分析差異表達(dá)基因所影響的代謝通路, 對(duì)前20個(gè)KEGG途徑進(jìn)行了富集分析。KEGG分析表明, α-亞麻酸代謝(alpha-linolenic acid metabolism)、不飽和脂肪酸生物合成(biosynthesis of unsaturated fatty acids)、氨基糖和核糖代謝(amino sugar and nucleotide sugar metabolism)、檸檬酸循環(huán)(citratecycle (TCA cycle))、甘油酯代謝(glycerolipid metabolism)、半乳糖代謝(galactose metabolism)顯著富集。前人研究表明長(zhǎng)鏈脂肪酸可以作為一種萌發(fā)抑制劑, 有可能是通過抑制胚胎生長(zhǎng)發(fā)揮作用[24]。另外, 許多氨基酸合成和代謝相關(guān)的條目, 如纈氨酸、亮氨酸和異亮氨酸合成(valine, leucine and isoleucine biosynthe- sis)、β-丙氨酸代謝(beta-alanine metabolism)、酪氨酸代謝(tyrosine metabolism)、苯丙氨酸、酪氨酸和色氨酸合成(phenylalanine, tyrosine and tryptophan biosynthesis)、苯丙氨酸代謝(phenylalanine metabolism)、精氨酸和脯氨酸代謝(arginine and proline metabolism)、酪氨酸代謝(tyrosine metabolism)、色氨酸代謝(tryptophan metabolism)也在HY52 vs M23 和HY52 vs M67的比較組中同時(shí)富集到(圖5)。除此之外, 蛋白外運(yùn)(protein export)和晝夜節(jié)律(circadian rhythm-plant)也在HY52 vs M67中顯著富集(圖5-F)。

2.5 花生休眠解除相關(guān)差異表達(dá)基因的富集分析

為揭示休眠解除機(jī)制, 將M23_12 vs M23_0, M23_24 vsM23_0, M67_12 vs M67_0, M67_24 vs M67_0比較組進(jìn)行了GO和KEGG富集分析(圖6和圖7)。KEGG富集分析結(jié)果表明, 差異表達(dá)基因富集在碳水化合物代謝相關(guān)的條目, 如甘油磷脂代謝(glycerophospholipid metabolism)、糖胺聚糖降解(glycosaminoglycan degradation)和氨基酸代謝相關(guān)的條目, 如苯丙氨酸、酪氨酸和色氨酸合成(phenylalanine, tyrosine and tryptophan biosynthesis)、甘氨酸、絲氨酸和蘇氨酸代謝(glycine, serine and threonine metabolism)、精氨酸和脯氨酸代謝(arginine and proline metabolism)。另外, 還富集到了檸檬酸循環(huán)(citrate cycle (TCA cycle))、蛋白外運(yùn)(protein export)、晝夜節(jié)律(circadian rhythm-plant)等條目(圖7)。而且在M23_24 vs M23_0比較組中, 富集到了質(zhì)子轉(zhuǎn)運(yùn)ATP酶活性(proton-transporting ATPase activity)和質(zhì)子轉(zhuǎn)運(yùn)ATP合酶活性(proton- transporting ATP synthase activity) 2個(gè)條目。

HY52: 花育52號(hào)。HY52: Huayu 52.

圖6 花生種子休眠解除過程中差異基因GO富集分析

圖7 花生種子休眠解除過程中差異基因KEGG富集分析

2.6 花生休眠維持和解除過程中與植物激素合成、代謝和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的差異表達(dá)基因

種子休眠和萌發(fā)是通過植物激素網(wǎng)絡(luò)調(diào)控, 包括ABA、GA、ETH和Auxin等, 植物激素合成、代謝和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的基因在種子休眠維持和解除過程中顯著表達(dá)。因?yàn)镸67中ABA含量和ABA/GA比值均低于M23 (圖1-E, F), 而且HY52 vs M67比HY52 vs M23有更多的差異表達(dá)基因(圖2-B), 因此以下分析以HY52 vs M67展示。

ABA 8’-羥化酶(CYP707A)是參與ABA代謝的關(guān)鍵酶, 有3個(gè)編碼CYP707A的基因在HY52和M67中都顯著上調(diào)(圖8)。9個(gè)ABA受體()基因隨著吸脹時(shí)間增加而上調(diào), ABA信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的34個(gè)(編碼蛋白磷酸酶2C)基因和4個(gè)基因也在吸脹過程中差異表達(dá)(圖8)。

GA合成相關(guān)基因, 如1個(gè)(編碼ent-貝殼杉酸氧化酶)和2個(gè)(編碼赤霉素20氧化酶)基因都在休眠解除過程中上調(diào), 但是參與GA失活的2個(gè)(編碼赤霉素2氧化酶)基因也上調(diào)表達(dá)(圖8)。此外, 1個(gè)赤霉素受體基因和2個(gè)信號(hào)轉(zhuǎn)導(dǎo)元件也參與這個(gè)過程。

乙烯是調(diào)控種子休眠解除的另一個(gè)關(guān)鍵植物激素, 外源施加乙烯能打破種子休眠[7]。乙烯合成基因(2個(gè))、(7個(gè))和5個(gè)乙烯受體基因包括, 都在HY52和M67吸脹過程中上調(diào)表達(dá)(圖8)。另外, 還有35個(gè)轉(zhuǎn)錄因子也在吸脹過程中有差異表達(dá)。生長(zhǎng)素合成和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的基因, 如1個(gè)(編碼吲哚-3-丙酮酸單加氧酶)、4個(gè)(編碼生長(zhǎng)素上調(diào)小RNA)和8個(gè)(編碼生長(zhǎng)素反應(yīng)因子)在HY52和M67的種子休眠解除過程中差異表達(dá)(圖8)。

2.7 花生休眠維持和解除過程中與碳水化合物和脂質(zhì)合成和代謝相關(guān)的差異表達(dá)基因

KEGG富集分析結(jié)果表明, 在HY52_12 vs M23_12中顯著富集了氨基糖和核糖代謝(amino sugar and nucleotides sugar metabolism)、α-亞麻酸代謝(alpha-linolenic acid metabolism) (圖5-C)條目, 在HY52_24 vs M23_24中顯著富集了不飽和脂肪酸合成(biosynthesis of unsaturated fattyacids) (圖5-D)條目。在HY52_24 vs M67_24中顯著富集了半乳糖代謝(galactose metabolism)、甘油酯代謝(glycerolipid metabolism)、不飽和脂肪酸合成(biosynthesis of unsaturated fatty) (圖5-F)條目。在M23_12 vs M23_0和M23_24 vs M23_0中分別富集了糖胺聚糖降解(glycosaminoglycan degradation)和甘油磷脂代謝(glycerophospholipid metabolism) (圖7-A, B)條目?；谝陨细患治鼋Y(jié)果, 對(duì)糖和脂質(zhì)代謝相關(guān)的基因進(jìn)行篩選, 挖掘到9個(gè)相關(guān)的基因在種子休眠維持和解除過程中發(fā)生表達(dá)變化(圖9)。葡萄糖-6-磷酸/磷酸轉(zhuǎn)運(yùn)蛋白(GPT)參與淀粉和葡萄糖轉(zhuǎn)化, 其編碼基因在種子休眠解除過程中上調(diào)(圖9-B)。參與脂質(zhì)合成的3-磷酸甘油?；D(zhuǎn)移酶(GPAT)基因在種子休眠解除過程中上調(diào), 參與脂質(zhì)代謝的3個(gè)長(zhǎng)鏈?；o酶A合酶(LACS)基因的表達(dá)量也在此過程中發(fā)生變化(圖9-A)。以上所有差異表達(dá)基因在M67中的表達(dá)量均高于HY52, 表明休眠種子更難以調(diào)動(dòng)能量?jī)?chǔ)備。

圖8 花生種子吸脹過程中與激素合成、代謝和信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的差異基因表達(dá)熱圖

A: ABA、GA、乙烯和生長(zhǎng)素的生物合成、代謝和信號(hào)轉(zhuǎn)導(dǎo)示意圖; B: 與植物激素相關(guān)的差異表達(dá)基因熱圖。HY52: 花育52號(hào)。

A: the diagram of biosynthesis, metabolism, and signal transduction in ABA, GA, ethylene, and auxin; B: the heatmaps of the relative expression patterns of DEGs related to plant hormones. HY52: Huayu 52.

圖9 花生種子吸脹過程中與碳水化合物、脂質(zhì)合成、代謝相關(guān)的差異基因表達(dá)熱圖

A: 脂質(zhì)代謝相關(guān)差異表達(dá)基因熱圖; B: 碳水化合物合成和代謝相關(guān)差異表達(dá)基因熱圖。HY52: 花育52號(hào)。

A: the relative expression patterns of DEGs related to lipid metabolism displayed by heatmap; B: the relative expression patterns of DEGs related to carbohydrate synthesis and metabolism displayed by heat map. HY52: Huayu 52; DEGs: differentially expressed genes.

3 討論

3.1 花生休眠維持和解除過程中植物激素網(wǎng)絡(luò)的調(diào)控

脫落酸和赤霉素是調(diào)節(jié)種子休眠狀態(tài)的2種主要激素, 它們通過調(diào)節(jié)激素含量和信號(hào)轉(zhuǎn)導(dǎo)的平衡發(fā)揮拮抗作用[7,25]。ABA維持種子休眠, 而GA促進(jìn)種子萌發(fā)。前人研究表明種子休眠是由較高的ABA/GA比值調(diào)控的, 休眠解除或萌發(fā)則是通過促進(jìn)GA合成和ABA降解導(dǎo)致ABA/GA比值下降來實(shí)現(xiàn)的[26-29]。綜上, GA負(fù)調(diào)控種子休眠而ABA正調(diào)控種子休眠。值得注意的是, 吸脹過程中, 雖然M23和M67中ABA/GA比值都顯著低于HY52, 但M67中ABA/GA比值更低, 暗示了M67的休眠性更弱。本研究中, GA含量在強(qiáng)休眠品種HY52和弱休眠種質(zhì)M23和M67中都隨著吸脹時(shí)間而增加(圖1-B)。但ABA含量在M23和M67中隨吸脹進(jìn)程而降低, 而且含量低于HY52 (圖1-C)。ABA/GA比值隨吸脹進(jìn)程降低, M23和M67中ABA/GA比值顯著低于HY52 (圖1-D)。盡管HY52中ABA/GA比值有大幅度下降, 但是其休眠狀態(tài)并未被打破, 暗示了HY52具有很強(qiáng)的休眠性, 解除休眠可能需要更低的ABA/GA比值。擬南芥和煙草中, ABA含量在吸脹過程中積累[26,30]。同時(shí), 在休眠解除過程中ABA代謝也會(huì)加快, 因此能測(cè)到的ABA含量是生物合成代謝后的凈值[27]。Lu14是一個(gè)休眠不深的花生品種, 在子葉吸脹4 h、16 h、28 h和52 h的過程中ABA/GA3比值從38.26極速降低到3.82[19]。本研究中, HY52、M23、M67中的ABA/GA比值分別從1335到676、986到651、676到343 (圖1-F), ABA/GA比值較高的原因可能是本研究中GA含量和ABA含量的單位不同。

GA生物合成基因的表達(dá)模式與GA含量變化一致(圖8-B), 除了植物激素合成相關(guān)的基因外, 信號(hào)轉(zhuǎn)導(dǎo)相關(guān)的蛋白也參與種子休眠解除[31-32]。ABA受體基因/在HY52和M67中上調(diào), 但是卻下調(diào)了(圖8-B)。在擬南芥中, PYL/PYR家族成員通過與ABA信號(hào)轉(zhuǎn)導(dǎo)途徑中的負(fù)調(diào)控因子PP2C互作發(fā)揮冗余功能[25]。據(jù)報(bào)道, 包括ABI1、ABI2在內(nèi)的多種PP2C參與了種子休眠調(diào)控[33]。GA信號(hào)轉(zhuǎn)導(dǎo)元件的突變也能影響種子萌發(fā), DELLA蛋白基因的功能缺失挽救了其不萌發(fā)的表型[34]。GA受體GID和下游蛋白DELLA也是種子休眠維持和解除的重要調(diào)控因子。與預(yù)期一致, 本研究中在弱休眠花生M67中上調(diào), 但是負(fù)調(diào)控因子則下調(diào)(圖10)。表明ABA和GA相關(guān)的差異表達(dá)基因在花生種子休眠維持和解除中發(fā)揮作用。

生長(zhǎng)素也是調(diào)控種子休眠維持和解除的重要植物激素, 已有報(bào)道表明, IAA參與ABA的互作從而促進(jìn)種子休眠[35-37]。在種子休眠解除過程中, 擬南芥、豌豆和小麥中IAA含量增加[38-40]。與前人研究結(jié)果一致, IAA信號(hào)相關(guān)基因, 如TIR1、IAA14和TCP均上調(diào)(圖10), 表明生長(zhǎng)素可能在花生種子休眠維持和解除中發(fā)揮作用。

3.2 花生休眠維持和解除過程中碳水化合物和氨基酸代謝與調(diào)控

種子萌發(fā)是一個(gè)耗能的過程, 三?；视?triacyl-glycerol, TAG)是花生主要的脂質(zhì)儲(chǔ)備物質(zhì)。在萌發(fā)過程中, TAG被脂肪酶水解為脂肪酸, 生成的脂肪酸被轉(zhuǎn)運(yùn)進(jìn)入過氧化物酶體經(jīng)β氧化和乙醛酸循環(huán)為萌發(fā)后的生長(zhǎng)過程提供能量和C骨架。

淀粉、油脂和蛋白質(zhì)等貯藏物質(zhì)是種子從休眠到發(fā)芽轉(zhuǎn)變的重要能源[19,41-42], 在種子休眠解除過程中, 淀粉、油脂和蛋白質(zhì)分別降解為可溶性糖、脂肪酸和氨基酸。雖然淀粉不是花生中的主要碳儲(chǔ)備, 但在種子休眠解除過程中, 參與淀粉和蔗糖轉(zhuǎn)化的5種GPT均上調(diào)(圖9-B)。油脂(三酰基甘油或TAG)和蛋白質(zhì)占花生種子含量的80%[43], 因此可能是主要的能量?jī)?chǔ)備。本研究結(jié)果表明, 參與脂質(zhì)生物合成和代謝的3個(gè)長(zhǎng)鏈?；o酶A合酶(LACS)基因可能參與種子休眠解除過程(圖9-A)。

圖10 HY52_12 vs M67_12 (A) and HY52_24 vs M67_24 (B)比較組前20個(gè)GO條目中與植物激素和脅迫相關(guān)的差異表達(dá)基因熱圖分析

HY52: 花育52號(hào)。HY52: Huayu 52.

與差異表達(dá)基因的結(jié)果一致, 與碳水化合物代謝相關(guān)的GO條目“阿拉伯糖生物合成過程”在HY52_12 vs M23_12 (圖4-A)和HY52_12 vs M67_12顯著富集(圖5-A); “半乳糖-蔗糖半乳糖基轉(zhuǎn)移酶活性”在HY52_24 vs M23_24 (圖4-B)和HY52_24 vs M67_24 (圖5-B)中顯著富集; “營(yíng)養(yǎng)庫活性”和“半乳糖代謝過程”分別在HY52_12 vs M23_12 (圖4-A)和HY52_12 vs M67_12 (圖5-A)顯著富集。與三?；视痛x相關(guān)的GO條目“三羧酸循環(huán)酶復(fù)合物”、“脂肪酸生物合成”在HY52_24 vs M67_24中顯著富集(圖5-B)。表明, 淀粉和油脂代謝在花生種子休眠解除中也可能發(fā)揮作用, 但是需要更多的實(shí)驗(yàn)數(shù)據(jù)支持。

本研究結(jié)果表明, 氨基酸代謝可能參與了花生種子的休眠, 花生種子富含蛋白質(zhì), 可降解為氨基酸, 表明蛋白質(zhì)可能是種子休眠解除和萌發(fā)的貯藏儲(chǔ)備, 與氨基酸生物合成和代謝相關(guān)的KEGG條目在HY52 vs M23和HY52 vs M67中顯著富集(圖7和圖8)。氨基酸不僅在大麥萌發(fā)后生物活性物質(zhì)(如細(xì)胞蛋白和分泌的水解酶)的生物合成中發(fā)揮重要作用[44-45], 而且是許多具有多功能的代謝物的前體[46]。Sato等[46]的研究表明, 主效休眠基因編碼丙氨酸轉(zhuǎn)氨酶(AlaAT)調(diào)控大麥種子休眠。

3.3 花生種子休眠維持和解除過程中的其他調(diào)控因子

值得注意的是, 在HY52 vs M23和HY52 vs M67比較組中還富集到了一些GO條目, 如“脅迫反應(yīng)” “活性氧代謝調(diào)控” “種子耐旱性獲得” (圖4和圖5)。在種子萌發(fā)過程中, 種子由相對(duì)靜止?fàn)顟B(tài)轉(zhuǎn)入活躍狀態(tài), 重新生長(zhǎng)的過程類似于脅迫響應(yīng)。大多數(shù)休眠種子在種子發(fā)育后期具有抗干燥性, 因此可以在干燥狀態(tài)下存活很長(zhǎng)時(shí)間。然而, 由于活性氧(ROS)的產(chǎn)生和氧化損傷的發(fā)生, 非休眠的種子并不耐干燥[7]。

此外, GO條目“晝夜節(jié)律的負(fù)調(diào)控”在HY52_12 vs M67_12中顯著富集(圖4-E), KEGG條目“晝夜節(jié)律”在HY52_24 vs M67_24中富集(圖5-F), 這表明晝夜節(jié)律也可能參與種子休眠調(diào)控。植物晝夜節(jié)律是內(nèi)源性產(chǎn)生和自我維持的特性, 因此它在恒定的環(huán)境條件下持續(xù)存在, 通常是恒定的光照(或黑暗)和恒定的溫度[47]。在擬南芥中, 生物鐘基因,()和()在整合環(huán)境信號(hào)控制休眠解除中發(fā)揮重要作用[48]。本研究結(jié)果表明, 休眠維持和解除的過程可能是晝夜節(jié)律的重新調(diào)整。

4 結(jié)論

本研究綜合分析了強(qiáng)休眠品種HY52和弱休眠種質(zhì)M23和M67在種子休眠維持和解除過程中的調(diào)控網(wǎng)絡(luò)。結(jié)果表明, 植物激素(脫落酸、赤霉素、乙烯和生長(zhǎng)素)的生物合成和信號(hào)轉(zhuǎn)導(dǎo)在調(diào)控種子休眠維持和解除中起關(guān)鍵作用。此外, 能量?jī)?chǔ)備(碳水化合物和脂質(zhì))、氨基酸代謝、晝夜節(jié)律也可能參與了種子休眠解除過程, 本研究拓展了對(duì)花生種子休眠維持和解除調(diào)控的認(rèn)知, 也為花生休眠育種奠定了理論基礎(chǔ)。

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Comparative transcriptome profiling of dormancy regulatory network in peanut

WANG Fei-Fei1, ZHANG Sheng-Zhong1, HU Xiao-Hui1, CHU Ye2, CUI Feng-Gao1, ZHONG Wen3, ZHAO Li-Bo4, ZHANG Tian-Yu3, GUO Jin-Tao5, YU Hao-Liang6, MIAO Hua-Rong1,*, and CHEN Jing1,*

1Shandong Peanut Research Institute, Qingdao 266100, Shandong, China;2Department of Horticulture, University of Georgia Tifton Campus, Tifton 31793, GA, United States;3Shandong Seed Administration Station, Jinan 250100, Shandong, China;4Qingdao Agricultural Technology Extension Center, Qingdao 266071, Shandong, China;5Agricultural Service Center of Hezhuang, Xinzheng, Zhengzhou 451150, Henan, China;6Yantai Fenglin Foodstuff Co., Ltd, Yantai 264108, Shandong, China

Seed dormancy is an important and complex agronomic trait affecting yield and quality of peanut (L.). Seed dormancy and germination was reported to be regulated by the balance between abscisic acid (ABA) and gibberellic acid (GA). In this study, transcriptomic sequencing was performed with Huayu 52 (HY52), a peanut cultivar with strong dormancy, and two EMS mutant lines from HY52 with a weak level of dormancy. Seeds from these three lines were imbibed for 0, 12, and 24 h before tissue harvesting and RNA seq analysis. GA content of M23 and M67 was significantly higher than HY52 at 12 h after imbibition, however, the ABA content and ABA/GA ratio were lower than HY52. A total of 31,374 differentially expressed genes (DEGs) including biosynthesis and signal transduction related genes of plant hormones such as ABA and GA were discovered. We identified 50 genes related to ABA, 8 genes related to GA, 49 genes related to ethylene, and 13 genes related to auxin. Expression profiles of ABA and GA related genes was consistent with the higher GA and lower ABA content in the mutants compared with HY52 after 12 h and 24 h imbibition. In addition, many DEGs involved in carbohydrate and lipid metabolism, amino acid metabolism, and glutathione metabolism pathway were also identified. There were 5 carbohydrate metabolism related genes () and 4 lipid metabolism related genes. In addition, differentially regulated circadian rhythm pathways were found to involve in the process of peanut seed dormancy release. These results suggested that the regulation of dormancy maintenance and release was more complicated than phytohormone balance.

peanut; dormancy maintenance; dormancy release; transcriptome; plant hormone; amino acid metabolism

2022-08-10;

2023-02-21;

2023-03-03.

10.3724/SP.J.1006.2023.24186

通信作者(Corresponding authors):陳靜, E-mail: mianbaohua2008@126.com: 苗華榮, E-mail: 1649813628@qq.com

E-mail: wangfeifeisj@163.com

本研究由國(guó)家自然科學(xué)基金青年基金項(xiàng)目(32001584, 32201876), 山東省自然科學(xué)基金面上項(xiàng)目(ZR2022MC045), 山東省農(nóng)業(yè)良種工程(2020LZGC001), 山東省農(nóng)業(yè)科學(xué)院創(chuàng)新工程(CXGC2022A03, CXGC2022A21), 青島市民生科技計(jì)劃項(xiàng)目(20-3-4-26-nsh)和新疆維吾爾自治區(qū)重大科技專項(xiàng)(2022A02008-3)資助。

This study was supported by the Youth Fund Project of the National Natural Science Foundation of China (32001584, 32201876), the General Project of Shandong Natural Science Foundation (ZR2022MC045), the Shandong Province Agriculture Improved Seed Project (2020LZGC001), the Innovation Project of Shandong Academy of Agriculture Sciences (CXGC2022A03, CXGC2022A21), the Qingdao People’s Livelihood Science and Technology Project (20-3-4-26-nsh), and the Major Science and Technology Program of Xinjiang Uygur Autonomous Region (2022A02008-3).

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This is an open access article under the CC BY-NC-ND license (http: //creativecommons.org/licenses/by-nc-nd/4.0/).