甘藍型油菜皖油20號種子不同部位油脂合成的轉(zhuǎn)錄調(diào)控分析

張宇婷 魯少平 金 誠 郭 亮

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甘藍型油菜皖油20號種子不同部位油脂合成的轉(zhuǎn)錄調(diào)控分析

張宇婷 魯少平 金 誠 郭 亮*

華中農(nóng)業(yè)大學作物遺傳改良國家重點實驗室, 湖北武漢 430070

甘藍型油菜是主要的油料作物之一, 種子含油量一般在35%~50%。油脂主要儲存于油菜種子胚中, 胚主要由子葉[包括外子葉(OC)和內(nèi)子葉(IC)和胚軸(EA)]組成。低芥酸油菜品種皖油20號(WY20)種子不同部位的含油量存在顯著差異。WY20的胚中, OC含油量最高, EA含油量最低。同時, 脂肪酸組成在種子不同部位也存在差異, EA中棕櫚酸(C16:0)、亞油酸(C18:2)及二十碳酸(C20:0)的比例均顯著高于子葉, 特別是C16:0在EA中的比例約為子葉的2倍。而油酸(C18:1)及二十碳烯酸(C20:1)在子葉中的比例均顯著高于EA。硬脂酸(C18:0)在OC中含量最低, 在IC和EA中無差別。亞麻酸(C18:3)則在OC中含量最高, 在IC和EA中無差異。對發(fā)育34d種子的IC、OC和EA進行轉(zhuǎn)錄組分析, 將三個部位中基因表達定量分析的結(jié)果兩兩比較后共發(fā)掘出7192個差異表達基因, 其中OC和IC之間差異表達基因數(shù)目較少, 子葉和EA間有較多的差異表達基因。子葉和胚軸中的差異表達基因富集在光合作用、脂肪酸代謝和葉綠素合成等生物學過程?；蚬δ茏⑨岋@示, 差異表達基因中有355個和脂質(zhì)代謝相關(guān), 且多集中在質(zhì)體中脂肪酸從頭合成途徑。本研究表明油脂合成途徑關(guān)鍵基因的差異調(diào)控是造成油菜種子不同部位含油量和脂肪酸組成差異的主要因素。

甘藍型油菜; 種子不同部位; 含油量; 脂肪酸組成; 轉(zhuǎn)錄調(diào)控

油菜是世界第三大油料作物, 也是我國第一大油料作物, 菜籽油是我國最主要的食用植物油來源之一[1]。油料作物的含油量是一項重要的生產(chǎn)指標, 是油料作物產(chǎn)量的重要因素之一。油菜種子含油量每提高1個百分點, 單位面積油菜的產(chǎn)油量就能提高2.3~2.5個百分點[2]。近年來, 我國油菜單產(chǎn)已達到世界平均水平, 但油菜種子中的油脂含量偏低, 導(dǎo)致國產(chǎn)菜籽油在國際市場缺乏競爭力且受到進口植物油的沖擊。因此, 提高油菜籽含油量對我國油菜產(chǎn)業(yè)發(fā)展極其重要[3-5]。油菜種子中各類脂肪酸的組成影響菜籽油的品質(zhì)及營養(yǎng)價值。脂肪酸根據(jù)結(jié)構(gòu)分為飽和脂肪酸和不飽和脂肪酸, 其中飽和脂肪酸(C16:0和C18:0等)熔點較高, 人體不易消化吸收, 易凝固在血管壁上。不飽和脂肪酸(C18:1、C18:2、C18:3等)熔點較低, 易被人體消化和吸收, 不易凝固或沉淀在血管壁上[6]。我國市場上銷售的食用植物油有很多種, 其中菜籽油的飽和脂肪酸的含量是所有植物油中最低的, 其油酸、亞油酸和亞麻酸含量比例合理, 是最健康的食用植物油之一[7]。

植物細胞中, 質(zhì)體和內(nèi)質(zhì)網(wǎng)是脂肪酸合成的主要場所。光合作用產(chǎn)生的糖類在細胞質(zhì)中經(jīng)糖酵解轉(zhuǎn)化為丙酮酸進入質(zhì)體, 丙酮酸在丙酮酸脫氫酶的作用下生成乙酰輔酶A, 此過程為脂肪酸從頭合成的第一步[8]。乙酰輔酶A再經(jīng)過一系列的縮合、還原及脫水反應(yīng)生成丁酰-酰基轉(zhuǎn)運蛋白, 完成脂肪酸鏈的第一次延伸[9]。接著, 酰基轉(zhuǎn)運蛋白繼續(xù)與乙酰輔酶A反應(yīng)并重復(fù)上述過程, 每反應(yīng)一次碳鏈增加2個碳原子, 直至碳鏈上碳原子數(shù)為16時停止延伸[10]。此時, 合成的16:0-ACP在酮脂酰基ACP合酶(KASII)和硬脂酰ACP脫氫酶(SAD)的作用下生成18:1-ACP。然后, 在?；?ACP硫脂酶(FATA和FATB)催化下, 脂肪酸從脂酰ACP脫離并在長鏈脂酰-CoA合成酶(LACS)作用下合成?；鵆oA[11]。?；鵆oA被轉(zhuǎn)運到內(nèi)質(zhì)網(wǎng)或胞質(zhì)中的?；鵆oA池中, 一部分酰基CoA經(jīng)3-磷酸甘油?；D(zhuǎn)移酶(GPAT)、溶血性磷脂酸?；D(zhuǎn)移酶(LPAAT)、磷脂酸磷酸酶(PAP)和二酰甘油轉(zhuǎn)酰酶(DGAT)途徑最后合成三?；视?TAG), 另一部分?；鵆oA經(jīng)溶血卵磷脂酰基轉(zhuǎn)移酶(LPCAT)作用將脂肪酸轉(zhuǎn)移到卵磷脂(PC)上, 經(jīng)過脂肪酸脫氫酶FAD2和FAD3對脂肪酸鏈修飾產(chǎn)生帶有多不飽和脂肪酸酸鏈的PC[12]。此時, PC在磷脂二酯酰甘油酰基轉(zhuǎn)移酶(PDAT)的催化下與二?；视?DAG)反應(yīng)生成TAG, 或者在磷脂酰膽堿: 二酰甘油磷酸膽堿轉(zhuǎn)移酶(PDCT)和磷酸膽堿轉(zhuǎn)移酶(CPT)的作用下生成DAG, 然后再合成TAG[13]。近年來研究表明, TAG和其他脂類分子在棉花、亞麻芥、油菜和擬南芥等種子中的含量是不均一的, 暗示脂質(zhì)代謝途徑在不同種子中存在著差異[14-17]。推測轉(zhuǎn)錄水平或酶活水平的調(diào)控是造成種子不同部位中脂質(zhì)代謝差異的原因, 然而具體的機制并不明確[18]。

隨著測序技術(shù)的發(fā)展, 轉(zhuǎn)錄組測序已廣泛應(yīng)用于生命科學的各個領(lǐng)域[19-20]。RNA測序(RNA-seq)能揭示在特定時間內(nèi)或部位中RNA的存在和數(shù)量, 是一種高通量的分析基因表達的手段[21-23]。Manuel等[24]基于深度表達序列標記測序?qū)?種油料作物種子的4個發(fā)展階段的基因表達進行轉(zhuǎn)錄組分析, 揭示了參與合成油脂的基因具有保守性和物種特異性。Ste?phane等[25]對油棕果實和種子的3個不同部位的轉(zhuǎn)錄組進行了比較, 發(fā)現(xiàn)EgWRI1-1和EgWRI1-2轉(zhuǎn)錄因子在果皮和胚乳的油脂積累過程中被大量轉(zhuǎn)錄。Lu等[26]比較高、低含油量油菜種子不同部位中脂質(zhì)代謝物與油脂合成相關(guān)基因表達, 結(jié)果表明種子不同部位的脂質(zhì)含量存在明顯差異, 這些差異主要由種子不同部位中參與油脂合成相關(guān)基因表達量的不同造成。但該研究沒有對造成種子不同部位脂質(zhì)含量差異的相關(guān)差異表達基因進行深入挖掘和分析。本研究從轉(zhuǎn)錄組水平分析并鑒定調(diào)控油菜種子不同部位含油量和脂肪酸組成的代謝網(wǎng)絡(luò)和關(guān)鍵基因, 從轉(zhuǎn)錄水平解析油菜種子不同部位中油脂合成的調(diào)控機制。

1 材料與方法

1.1 材料及處理方法

供試材料為甘藍型低芥酸油菜皖油20 (WY20)種子。顯微鏡下分離WY20發(fā)育34 d種子的內(nèi)子葉(IC)、外子葉(OC)、胚軸(EA)和種皮(SC), 設(shè)置5個生物學重復(fù)。參考Lu等[26]的方法用脂肪酸甲酯化和氣質(zhì)聯(lián)用儀(GC-MS)分析種子各部位的油脂含量和脂肪酸組成。提取IC、OC和EA總RNA進行轉(zhuǎn)錄組測序, 設(shè)每組3個生物學重復(fù)。

1.2 文庫構(gòu)建與測序

使用植物RNA提取試劑盒(DP432, http://www. tiangen.com/)提取種子各個部位的總RNA, 將提取的RNA樣品送GenoSeq公司(http://www.genoseq. cn/), 用Illumina Hiseq進行轉(zhuǎn)錄組測序, 每個樣品6G數(shù)據(jù)量。

1.3 轉(zhuǎn)錄組數(shù)據(jù)分析

用fastp軟件去除接頭序列和低質(zhì)量讀數(shù), 測序數(shù)據(jù)質(zhì)量控制和過濾后, 通過hisat2軟件將9組轉(zhuǎn)錄組數(shù)據(jù)比對到油菜參考基因組(http://www.genoscope. cns.fr/brassicanapus/)。利用featureCounts軟件定量得到TPM值(transcripts per kilobase of exon model per million mapped reads)來計算基因的表達量[27-28]。再用DESeq2軟件包進行差異表達基因的篩選, 篩選條件為-value <0.01, |log2fold change|> 1, 并對所篩選出的差異表達基因進行GO富集分析[29-30]。

1.4 候選基因的鑒定及篩選

利用blastp工具將油菜蛋白序列與數(shù)據(jù)庫中擬南芥蛋白序列對比, 設(shè)置E-value為1e–5, Coverage> 50%, 找到油菜在擬南芥中對應(yīng)的同源基因及基因ID。利用擬南芥約700個與脂質(zhì)代謝相關(guān)的基因和甘藍型油菜全基因組測序得到的1000余個與油脂相關(guān)的基因作為參考, 進行油菜基因同源性分析從而構(gòu)建本研究中油菜油脂合成基因數(shù)據(jù)庫。將差異表達分析的結(jié)果與數(shù)據(jù)庫相結(jié)合, 篩選出與油脂合成相關(guān)的差異表達基因, 進一步對油脂合成相關(guān)的差異表達基因進行功能注釋和表達數(shù)據(jù)的分析。最后將與油脂合成相關(guān)的差異表達基因比對到油脂合成的代謝途徑中, 并在代謝途徑中標記出差異基因在IC、OC和EA兩兩之間表達量差異的倍數(shù)。

2 結(jié)果與分析

2.1 WY20種子不同部位脂肪酸組成和含油量分析

WY20種子切片在顯微鏡下可以明顯地看到OC、IC、EA和SC四個不同部位(圖1-A)。脂肪酸分析結(jié)果表明, 不同部位之間含油量存在顯著差異, OC的含油量最高, SC的含油量最低(圖1-B)。比較種子不同部位所占種子重量的百分比, 含油量最高的OC的比例遠大于其他3個部位, 含油量最少的SC比例高于EA (圖1-C)。WY20種子不同部位的脂肪酸組成存在顯著差異, 其中C18脂肪酸在整顆種子中的脂肪酸含量比較高(圖1-D, E)。C16:0、C18:2和C20:0在胚軸中的含量均顯著高于子葉, 特別是C16:0在EA中的含量約為子葉的2倍。C18:1和C20:1在子葉中的含量則均顯著高于EA, C18:0在OC中含量顯著低于IC和EA, 而在IC和EA中無差別。C18:3則在OC中含量最高, 在IC和EA中無差異(圖1-D)。

2.2 轉(zhuǎn)錄組質(zhì)量控制及reads的比對

數(shù)據(jù)統(tǒng)計約有3.08億條原始數(shù)據(jù), 平均每個部位有1.02億條。經(jīng)質(zhì)量控制和過濾后, 得到質(zhì)量較好的讀段(reads), reads和全基因組比對有93.87%被映射到油菜參考基因組?？傋x數(shù)中約有6.13%的reads可能由于篩選參數(shù)設(shè)定較嚴格、測序組裝錯誤或者參考基因組不完整而匹配不上。

2.3 基因差異表達分析

調(diào)查3個部位中基因表達量的分布并對樣品3個生物學重復(fù)之間的相關(guān)性進行檢測和主成分分析(PCA)(圖2-A)表明, IC和OC三個生物學重復(fù)之間的皮爾森系數(shù)均高于0.98, EA三個生物學重復(fù)之間的皮爾森系數(shù)高于0.92, 但生物學重復(fù)之間差異均不顯著, 證明3個生物學重復(fù)的重復(fù)性較好。對3個部位的基因表達量進行兩兩間的差異分析表明, IC和OC間比較得到了525個差異基因, 其中有233個基因表達上調(diào), 292個表達下調(diào)。IC和EA間比較得到了5436個差異基因, 表達上調(diào)的有2520個, 下調(diào)的有2916個。OC和EA間比較得到了5749個差異基因, 表達上調(diào)的有2535個, 下調(diào)的有3214個(圖2-B)。3個組合去重復(fù)后的差異表達基因有7192個, 有116個基因在EA與IC, EA與OC, IC與OC 3個比較組合中均有表達上的差異(圖2-C)。此結(jié)果也表明內(nèi)子葉和外子葉間差異基因較少, 暗示著OC和IC的基因表達模式比較一致。

2.4 GO富集分析

GO富集分析發(fā)現(xiàn), OC與EA和IC與EA之間的差異基因主要富集在光合作用、一元羧酸生物合成過程和脂肪酸生物合成過程。子葉與EA間存在較多參與脂質(zhì)合成相關(guān)的差異表達基因, 這些基因?qū)⒊蔀槲覀兒罄m(xù)分析的重點。而OC與IC中的差異表達基因很少, 且主要富集在胞外區(qū)和核仁等與油脂合成不直接相關(guān)的生物學過程。我們列出了每組比較差異表達基因GO富集到的前10個代謝通路(圖3)。

圖1 甘藍型油菜WY20種子不同部位含油量和脂肪酸組成

A: 顯微鏡明視野下WY20種子部位; B: 種子不同部位含油量; C: 種子不同部位重量百分比; D: 種子不同部位脂肪酸組成; E: 整顆種子脂肪酸組成。

A: bright-field image in different parts of WY20 seed under microscope; B: oil content in different parts of seed; C: weight percentage in different parts of seed; D: fatty acid composition in different parts of seed; E: whole seed fatty acid composition.

圖2 3個種子部位差異表達基因分布情況

A: 樣本間PCA分析; B: IC、OC和EA差異表達基因數(shù)目及百分比; C:差異表達基因維恩圖。

A: PCA analysis of different samples; B: differentially expressed genes in IC, OC, and EA; C: Venn diagram of differentially expressed genes.

2.5 候選基因的鑒定與篩選

在油菜種子不同部位之間篩選的7192個差異表達基因中, IC與OC之間的差異表達基因中有18個油脂合成相關(guān)基因, IC與EA之間的差異表達基因中有268個油脂合成相關(guān)基因, OC與EA之間的差異表達基因中有286個油脂合成相關(guān)基因。去除相互之間重復(fù)基因, 一共篩選出355個和油脂合成相關(guān)的差異基因。為了確保差異基因在種子中有表達, 去除這些基因的表達量(TPM值)在3個部位中均小于1的基因, 最終剩余336個基因。再將336個基因匹配到油菜種子油脂合成途徑中, 共得到53個可以匹配到代謝通路上的基因, 我們將同一基因的不同拷貝算做1類基因。IC與OC間得到了4類基因, IC與EA間得到了49類基因, OC與EA間得到了46類基因。其中BnaA02g11570D、BnaA03g23490D、BnaA03g13590D和BnaC03g27860D為3種組合對比中共有的差異基因。而IC與EA、OC與EA兩種對比組合間共有匹配到通路上的和油脂合成相關(guān)的差異表達基因有38個, IC與EA間特有7個, OC與EA間特有4個。我們將得到的53個基因根據(jù)不同對比組合進行了注釋和分析(圖4和表1)。油脂合成途徑中IC和OC中的基因表達量都普遍高于EA, 而IC和OC之間的基因表達水平差別不顯著(圖4)。

圖3 油菜胚的3個部位差異表達基因GO功能分布

A: IC和OC差異表達基因功能分布; B: IC和EA差異表達基因功能分布; C: OC和EA差異表達基因功能分布。

A: GO analysis of differentially expressed genes between IC and OC; B: GO analysis of differentially expressed genes between IC and EA; C: GO analysis of differentially expressed genes between OC and OC.

3 討論

植物種子中的油脂合成過程是一個復(fù)雜的網(wǎng)絡(luò), 發(fā)生在細胞的多個細胞器中, 受多種酶、代謝物轉(zhuǎn)運、轉(zhuǎn)錄因子和能量代謝等影響[31]。近年來研究表明, 脂質(zhì)代謝途徑在油料植物種子胚的不同部位存在著差異[14-17]。本研究對低芥酸油菜WY20種子不同部位的分析結(jié)果表明, 含油量在IC、OC和EA中存在著顯著差異, OC含油量最高, IC含油量次之, EA含油量最低。C16:0和C18:2在EA中的比例顯著高于IC和OC, 而C18:3在EA中比例最低(圖1-B, C)。本研究結(jié)果與前人利用不同的油菜材料研究結(jié)果基本一致[26], 表明油菜種子不同部位中油脂合成和脂肪酸脫飽和過程受到不同的調(diào)控。Borisjuk等[32]研究表明油菜種子不同部位能量代謝有明顯差異, 這可能與油菜種子結(jié)構(gòu)有關(guān), 不同種子部位獲得的空間和光不同, 導(dǎo)致光合作用等存在著差別。

圖4 油脂合成相關(guān)差異表達基因在油脂代謝通路中的比較分析

維恩圖中陰影代表差異表達基因的分布。紅色數(shù)字表示蛋白家族對應(yīng)基因TPM值的平均值在IC和EA間的比值, 綠色數(shù)字表示蛋白家族對應(yīng)基因TPM值的平均值在OC和EA間的比值, 藍色數(shù)字表示蛋白家族對應(yīng)基因TPM值的平均值在IC和OC間的比值。

編碼蛋白質(zhì)的基因: DHLAT: 二氫硫辛酰胺乙酰轉(zhuǎn)移酶; LPD: 二氫硫辛酰胺脫氫酶; BCCP: 生物素羧基載體蛋白; ACCase: 乙酰輔酶a羧化酶; MCMT: ?；d體蛋白; KASI: 3-酮?；?酰基載體蛋白合酶I; KASII: 3-酮?；?酰基載體蛋白合酶II; KASIII: 3-酮?；?酰基載體蛋白合酶III; KAR: 酮脂酰還原酶; HAD: 硫酯酶蛋白; ENR: 烯酰ACP還原酶; ACP: ?；d體蛋白; SAD: 硬脂酰-?；d體蛋白脫飽和酶蛋白; FATA: FATA硫酯酶; FATB: FATB硫酯酶; LACS9: 長鏈?；o酶9; FAD2: 脂肪酸去飽和酶2; FAD3: 脂肪酸去飽和酶3; CPT: CDP膽堿-甘油二酯膽堿酯酶; PDCT: 磷脂酰膽堿-甘油二酯膽堿酯酶; GPAT: 磷酸甘油脂酰轉(zhuǎn)移酶; PDAT: 磷脂-二酰甘油?；D(zhuǎn)移酶; LPAAT: 溶血磷脂酸酰基轉(zhuǎn)移酶; PAP: 磷脂酸磷酸酶; DGAT: 二酰甘油?；D(zhuǎn)移酶; OBO: 油體蛋白; LTP: 脂質(zhì)轉(zhuǎn)運蛋白; ER: 內(nèi)質(zhì)網(wǎng)。

The shadow in venn diagram represents where the differentially expressed genes are located. Number in red indicates the ratio of genes’ average TPM of between IC and EA, number in green indicates the ratio of genes’ average TPM of between OC and EA, number in blue indicates the ratio of genes’ average TPM of between IC and OC.

Abbreviation of genes that encode proteins: DHLAT: dihydrolipoamide acetyltransferase; LPD: dihydrolipoamide dehydrogenase; BCCP: biotin carboxyl carrier protein; ACCase: acetyl-CoA carboxylase; MCMT: malonyl-CoA: ACP malonyltransferase; KASI: 3-ketoacyl-acyl carrier protein synthase I; KASII: 3-ketoacyl-acyl carrier protein synthase II; KASIII: 3-ketoacyl-acyl carrier protein synthase III; KAR: ketoacyl-ACP reductase; HAD: hydroxyacyl-ACP dehydrase; ENR: enoyl-ACP reductase; ACP: acyl carrier protein; SAD: stearoyl-acyl carrier protein desaturase; FATA: acyl-ACP thioesterase A; FATB: acyl-ACP thioesterase B; LACS: long-chain acyl-CoA synthetase; FAD2: FA desaturase 2; FAD3: FA desaturase 3; CPT: CDP-choline: diacylglycerol cholinephosphotransferase; PDCT: phosphatidylcholine:diacylglycerol cholinephosphotransferase; GPAT: glycerol-3-phosphate acyltransferase; PDAT: phospholipid:diacylglycerol acyltransferase; LPAAT: lysophosphatidic acid acyltransferase; PAP: phosphatidic acid phosphatase; DGAT: diacylglycerol acyltransferase; OBO: oil body oleosin; LTP: lipid transfer protein; ER: endoplasmic reticulum.

表1 甘藍型油菜皖油20種子不同部位油脂合成差異表達基因

(續(xù)表1)

基因名Gene ID注釋描述Annotation description蛋白家族縮寫Protein family abbreviations分組Group BnaC03g27860DHydroxysteroid dehydrogenase 1OBOEA-IC-OC BnaCnng57830DHydroxysteroid dehydrogenase 1OBOEA-IC BnaA09g02110DOleosinOBOEA-OC BnaA03g20420DStearoyl-acyl-carrier-protein desaturase proteinSADEA-IC/EA-OC BnaA01g32860DStearoyl-acyl-carrier-protein desaturase proteinSADEA-IC/EA-OC BnaC03g24420DStearoyl-acyl-carrier-protein desaturase proteinSADEA-IC/EA-OC BnaC09g41580DStearoyl-acyl-carrier-protein desaturase proteinSADEA-IC/EA-OC BnaA10g18080DStearoyl-acyl-carrier-protein desaturase proteinSADEA-IC/EA-OC BnaA05g03490DStearoyl-acyl-carrier-protein desaturaseSADEA-OC BnaC04g03030DStearoyl-acyl-carrier-protein desaturaseSADEA-OC BnaC09g19280D3-ketoacyl-acyl carrier protein synthase IKASIEA-IC/EA-OC BnaA02g24400D3-ketoacyl-acyl carrier protein synthase IKASIEA-IC/EA-OC BnaA06g36060D3-ketoacyl-acyl carrier protein synthase IKASIEA-IC/EA-OC BnaC06g35760D3-ketoacyl-acyl carrier protein synthase IIKAS IIEA-IC/EA-OC BnaA07g31890D3-ketoacyl-acyl carrier protein synthase IIKAS IIEA-IC/EA-OC BnaA07g21940D3-ketoacyl-acyl carrier protein synthase IIKAS IIEA-IC/EA-OC BnaC06g22680D3-ketoacyl-acyl carrier protein synthase IIKAS IIEA-IC BnaA04g07120DAcyl-ACP thioesteraseFATAEA-IC/EA-OC BnaCnng41490DAcyl-ACP thioesteraseFATAEA-IC/EA-OC BnaCnng00070DFATA acyl-ACP thioesterase FATAFATAEA-IC/EA-OC BnaA07g05070DFATA acyl-ACP thioesterase FATAFATAEA-IC/EA-OC BnaC03g75820DKetoacyl-ACP ReductaseKAREA-IC/EA-OC BnaA02g13310DBeta-ketoacyl reductaseKAREA-IC/EA-OC BnaA07g26670DBeta-ketoacyl reductaseKAREA-IC/EA-OC BnaC06g28830DBeta-ketoacyl reductaseKAREA-IC/EA-OC BnaC09g16320DAcyl carrier proteinACPEA-IC/EA-OC BnaC09g03000DAcyl carrier proteinACPEA-IC/EA-OC BnaA09g03610DAcyl carrier proteinACPEA-IC/EA-OC BnaAnng23710DAcyl carrier proteinACPEA-OC BnaC03g45040DEnoyl-ACP ReductaseENREA-IC/EA-OC BnaC07g04330DEnoyl-ACP ReductaseENREA-IC/EA-OC BnaA03g38220DEnoyl-ACP ReductaseENREA-IC/EA-OC BnaAnng02240DE2 component of pyruvate dehydrogenase complexDHLATEA-IC/EA-OC BnaC06g08280DE2 component of pyruvate dehydrogenase complexDHLATEA-IC/EA-OC BnaC07g23030DE2 component of pyruvate dehydrogenase complexDHLATEA-IC BnaA06g33300DE2 component of pyruvate dehydrogenase complexDHLATEA-IC BnaAnng22560DChloroplasticacetyl coenzyme A carboxylaseBCCPEA-IC/EA-OC BnaC09g42420DBiotin carboxyl carrier protein 2BCCPEA-IC/EA-OC BnaA03g02830DThioesterase superfamily proteinHADEA-IC/EA-OC BnaA02g00390DThioesterase superfamily proteinHADEA-IC BnaA07g20920DLong chain acyl-CoA synthetase 9LACS9EA-IC/EA-OC BnaC06g20910DLong chain acyl-CoA synthetase 9LACS9EA-IC BnaA01g17630DE3 component of pyruvate dehydrogenase complexLPDEA-IC/EA-OC BnaCnng75250DAcetyl-CoA carboxylaseACCaseEA-IC BnaA05g12180DAcyl-carrier-proteinMCMTEA-IC/EA-OC

Lu等[26]對ZS11和WH5557兩個油菜材料的種子不同部位進行分析表明, IC、OC和EA中脂質(zhì)代謝物含量存在顯著差異, IC、OC和EA中糖酵解途徑和油脂合成途徑的基因表達水平普遍存在著顯著差異。并進一步鑒定了編碼LPAAT、PAP、DGAT、Oleosin等調(diào)控種子不同部位脂質(zhì)含量差異的關(guān)鍵基因。本研究在前期工作基礎(chǔ)上, 對低芥酸油菜WY20種子不同部位進行轉(zhuǎn)錄組分析, 進一步從轉(zhuǎn)錄水平解析油菜種子不同部位含油量和脂肪酸差異的調(diào)控機制。WY20種子3個部位轉(zhuǎn)錄組分析結(jié)果顯示, EA與IC和OC之間的差異表達基因主要富集在光合作用、一元羧酸合成和脂肪酸合成與代謝過程, 這些差異表達基因可能是造成不同部位含油量和脂肪酸差異的主要因素(圖3-B, C)。IC和OC之間差異表達基因僅有18個基因與脂質(zhì)代謝相關(guān), 而EA與IC和OC之間與脂質(zhì)代謝相關(guān)的差異表達基因分別為268個和286個, 與EA含油量和脂肪酸組成與IC和OC差異比較一致(圖1-B, D)。對油脂合成途徑進行深入分析顯示, IC和OC中與油脂合成相關(guān)的基因的表達量普遍是EA的2~3倍, 例如在IC和OC中的表達量分別是EA中的2.8倍和2.5倍(圖4)。這些差異表達基因主要集中在質(zhì)體中脂肪酸合成途徑, 因此我們推斷這些關(guān)鍵基因在轉(zhuǎn)錄水平的調(diào)控是造成EA含油量比IC和OC低的主要原因(圖4)。

脂肪酸組成主要由脂肪酸脫飽和酶和脂肪酸延長酶調(diào)控[33]。植物質(zhì)體中由SAD催化合成18:1-ACP, 而其余不飽和脂肪酸合成的反應(yīng)主要發(fā)生在內(nèi)質(zhì)網(wǎng)中。從質(zhì)體轉(zhuǎn)運出來的18:1-CoA被合成PC, 接著在油酸去飽和酶(FAD2)和亞油酸去飽和酶(FAD3)作用下分別生成18:2-PC和18:3-PC。本研究結(jié)果表明在IC和OC中表達量分別為EA的2.4倍和2.2倍, 與EA中C18:1比例最低而C18:2比例最高結(jié)果一致。在IC和OC中表達量分別為EA的3.8倍和4.1倍, 與EA中C18:3比例最低結(jié)果一致。而EA中C16:0的含量約為IC和OC的2倍, 從代謝途徑看,、和在IC和OC中的表達量均顯著高于EA (2.5~3.7倍), 因此C16:0在IC和OC中被更高效地轉(zhuǎn)化為C18:1, 這與IC和OC中C16:0比例比EA低一致(圖1-D和圖4)。說明、、、和等關(guān)鍵基因在WY20種子的表達有部位特異性, 這些基因的表達量決定了種子不同部位中脂肪酸的組成。

4 結(jié)論

WY20油菜種胚不同部位的含油量和脂肪酸組成存在著差異, 尤其是EA的含油量最低并且脂肪酸組成與子葉存在較大差異。EA與子葉之間參與油脂合成的差異表達基因較多。EA中參與質(zhì)體中脂肪酸合成的基因表達量普遍低于子葉, 造成EA含油量比子葉低。EA中、、、和等關(guān)鍵基因表達量顯著低于子葉, 造成EA中C16:0和C18:2高于子葉。轉(zhuǎn)錄調(diào)控是WY20油菜種子不同部位油脂合成差異的主要機制, 該研究對于理解油菜種子不同部位含油量和脂肪酸組成差異具有一定的科學意義, 對油菜和其他油料作物高含油量育種和品質(zhì)改良具有重要的指導(dǎo)意義。

[1] 沈金雄, 傅廷棟. 我國油菜生產(chǎn)、改良與食用油供給安全. 中國農(nóng)業(yè)科技導(dǎo)報, 2011, 13(1): 1–8 Shen J X, Fu T D. Rapeseed production improvement and edible oil supply in China., 2011, 13(1): 1–8 (in Chinese with English abstract)

[2] 沈瓊. 中國油菜產(chǎn)業(yè)競爭優(yōu)勢與劣勢分析. 農(nóng)業(yè)產(chǎn)品加工, 2008, (8): 57–59 Shen Q. Analysis on competitive advantages and disadvantages of Chinese rapeseed industry., 2008, (8): 57–59 (in Chinese with English abstract)

[3] 李殿榮, 田建華, 陳文杰, 張文學, 李永紅, 王灝. 甘藍型油菜特高含油量育種技術(shù)與資源創(chuàng)新. 西北農(nóng)業(yè)學報, 2011, 20(12): 83–87 Li D R, Tian J H, Chen W J, Zhang W X, Li Y H, Wang H. Breeding technologies and germplasm innovation on extra- high-oil content in., 2011, 20(12): 83–87 (in Chinese with English abstract)

[4] 張永霞, 趙鋒, 張紅玲.中國油菜產(chǎn)業(yè)發(fā)展現(xiàn)狀、問題及對策分析. 世界農(nóng)業(yè), 2015, (4): 96–99 Zhang Y X, Zhao F, Zhang H L. Analysis on the development status, problems and countermeasures of Chinese rapeseed industry., 2015, (4): 96–99 (in Chinese with English abstract)

[5] 王漢中, 殷艷. 我國油料產(chǎn)業(yè)形勢分析與發(fā)展對策建議. 中國油料作物學報, 2014, 36: 414–421 Wang H Z, Yin Y. Analysis and strategy for oil crop industry in China., 2014, 36: 414–421 (in Chinese with English abstract)

[6] 熊秋芳, 張效明, 文靜, 李興華, 傅廷棟, 沈金雄. 菜籽油與不同食用植物油營養(yǎng)品質(zhì)的比較——兼論油菜品質(zhì)的遺傳改良. 中國糧油學報, 2014, 29: 122–128 Xiong Q F, Zhang X M, Wen J, Li X H, Fu T D, Shen J X. Comparison of nutritive quality between rapeseed oil and different edible vegetable oil—on the genetic improvement of rapeseed quality., 2014, 29: 122–128 (in Chinese with English abstract)

[7] 熊源. 植物油的種類與營養(yǎng)價值. 中國糧食經(jīng)濟, 2014, (6): 72 Xiong Y. Types and nutritional value of vegetable oil., 2014, (6): 72 (in Chinese with English abstract)

[8] Saha S, Enugutti B, Rajakumari S. Cytosolic triacylglycerol biosynthetic pathway in oilseeds. Molecular cloning and expression of peanut cytosolic. diacylglycerol acyltransferase., 2006, 141: 1533–1543

[9] Thelen J J, Ohlrogge J B. Metabolic engineering of fatty acid biosynthesis in Plants., 2002, 4: 12–21

[10] Dahlqvist A, Stahl U, Lenman M. Phospholipid: diacylglycerol acyltransferase: An enzyme that catalyzes the Acyl-CoA- Independent formation of triacylglycerol in yeast and plants., 2000, 97: 6487–6492

[11] 周奕華, 陳正華. 植物種子中脂肪酸代謝途徑的遺傳調(diào)控與基因工程. 植物學通報, 1998, 15(5): 16–23 Zhou Y H, Chen Z H. Genetic manipulation and gene engineering of fatty acid metabolism in plant seeds., 1998, 15(5): 16–23 (in Chinese with English abstract)

[12] 周丹, 趙江哲, 柏楊, 張群, 井文, 章文華. 植物油脂合成代謝及調(diào)控的研究進展. 南京農(nóng)業(yè)大學學報, 2012, 35(5): 81–90 Zhou D, Zhao J Z, Bai Y, Zhang Q, Jing W, Zhang W H. Research advance in triacylglycerol synthesis, metabolism and regulation in plants. 2012, 35(5): 81–90 (in Chinese with English abstract)

[13] Bates P D, Stymne S, Ohlrogge J B. Biochemical pathways in seed oil synthesis., 2013, 16: 358–364

[14] Horn P J, Korte A R, Neogi P B, Love E, Fuchs J, Strupat K, Borisjuk L, Shulaev V, Lee Y J, Chapman K D. Spatial mapping of lipids at cellular resolution in embryos of cotton., 2012, 24: 622–636

[15] Horn P J, Silva J E, Anderson D, Fuchs J, Borisjuk L, Nazarenus T J, Shulaev V, Cahoon E B, Chapman K D. Imaging heterogeneity of membrane and storage lipids in transgenicseeds with altered fatty acid profiles., 2013, 76: 138–150

[16] Sturtevant D, Due?as M E, Lee Y J, Chapman K D. Three- dimensional visualization of membrane phospholipid distributions inseeds: a spatial perspective of molecular heterogeneity., 2017, 1862: 268 –281

[17] Woodfield H K, Sturtevant D, Borisjuk L, Munz E, Guschina I A, Chapman K, Harwood J L. Spatial and temporal mapping of key lipid species inseeds., 1998, 173: 1998–2009

[18] Sturtevant D, Lee Y J, Chapman K D. Matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) for direct visualization of plant metabolites in situ., 2015, 37: 53–60

[19] Lu X, Chen D, Shu D. The differential transcription network between embryo and endosperm in the early developing maize seed., 2013, 162: 440–455

[20] He R, Salvato F, Park J J. A systems-wide comparison of red rice () tissues identifies rhizome specific genes and proteins that are targets for cultivated rice improvement., 2014, 14: 46–66

[21] Schuster S C. Next-generation sequencing transforms today’s biology., 2008, 5: 16–18

[22] Metzker M L. Sequencing technologies: the next generation., 2010, 11: 31–46

[23] Louisa F L. RNA-Seq: a revolutionary tool for transcriptomics., 2008, 9: 568–574

[24] Troncoso-Ponce M A, Kilaru A, Cao X, Durrett T P, Fan J, Jensen J K, Thrower N A, Pauly M, Wilkerson C, Ohlrogge J B. Comparative deep transcriptional profiling of four developing oil seeds., 2011, 68: 1014–1027

[25] Dussert S, Morcillo F. Comparative transcriptome analysis of three oil palm fruit and seed tissues that differ in oil content and fatty acid composition., 2013, 162: 1337–1358

[26] Lu S P, Sturtevant D, Aziz M, Jin C, Li Q, Chapman K D, Guo L. Spatial analysis of lipid metabolites and expressed genes reveals tissue-specific heterogeneity of lipid metabolism in high- and low-oilL. seeds., 2018, 94: 915–932

[27] Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley D R, Pimentel H, Salzberg S L, Rinn J L, Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks., 2012, 7: 562–578

[28] Anders S, Pyl P T, Huber W. HTSeq: a Python framework to work with high-throughput sequencing data., 2015, 31: 166–169

[29] Wang L, Feng Z, Wang X, Wang X, Zhang X. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data., 2010, 26: 136–138

[30] Love M I, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2., 2014, 15: 550–575

[31] Baud S, Lepiniec L. Physiological and developmental regulation of seed oil production., 2010, 49: 235–249

[32] Borisjuk L, Neuberger T, Schwender J, Heinzel N, Sunderhaus S, Fuchs J, Hay J O, Tschiersch H, Braun H P, Denolf P, Lambert B, Jakob P M, Rolletschek H. Seed architecture shapes embryo metabolism in oilseed rape., 2013, 25: 113–128

[33] Napier J A, Haslam R P, Beaudoin F. Understanding and manipulating plant lipid composition: Metabolic engineering leads the way., 2014, 19: 68–75

Transcriptional regulation of oil biosynthesis in different parts of Wanyou 20 () seeds

ZHANG Yu-Ting, LU Shao-Ping, JIN Cheng, and GUO Liang*

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China

is one of the main oil crops and the seed oil content is generally between 35% and 50%. Oil is mainly stored in the seed embryo. Embryo is composed of cotyledons (including outer and inner cotyledons) and embryonic axis. The oil content and fatty acid composition of different parts of low erucicWY20’s seed were analyzed. There was a significant difference in oil content in different parts of the seed. The oil content in the outer cotyledon was the highest while embryonic axis had the lowest oil content. At the same time, the fatty acid composition also showed significant difference in different parts of the seed. The ratio of C16:0, C18:2, and C20:0 in embryonic axis was significantly higher than that in cotyledon. The ratio of C16:0 in the embryonic axis was about twice more than that of the cotyledons. The ratio of C18:1 and C20:1 in cotyledons was significantly higher than that in embryonic axis. C18:0 had the lowest content in the outer cotyledon and no difference in the inner cotyledons and embryonic axis. C18:3 had the highest content in the outer cotyledons and no difference between inner cotyledons and embryonic axis. Transcriptome analysis was performed for the inner cotyledon, outer cotyledon and embryonic axis of the 34-day-old seed. A total of 7192 differentially expressed genes (DEGs) were identified after pairwise comparison of gene expression of the three parts. There were much fewer DEGs between cotyledons and more DEGs between cotyledon and embryonic axis. These DEGs were enriched in biological processes such as photosynthesis, fatty acid metabolism and chlorophyll metabolism. Gene function annotations revealed that there were 355 genes involved in lipid metabolism, especially in the de novo fatty acid biosynthesis in plastid. This study suggests that transcriptional regulation of key genes involved in oil biosynthesis results in different oil contents and fatty acid compositions in different parts of seed in.

; different seed parts; oil content; fatty acid composition; transcriptional regulation

2018-07-28;

2018-12-24;

2019-01-03.

10.3724/SP.J.1006.2019.84105

郭亮, E-mail: guoliang@mail.hzau.edu.cn

E-mail: 249749481@qq.com

本研究由國家自然科學基金青年科學基金項目(31701458)和中央高?；究蒲袠I(yè)務(wù)費專項資金資助項目(2662015PY090)資助。

This study was supported by the National Science Foundation for Young Scientists of China (31701458) and Fundamental Research Funds for the Central Universities (2662015PY090).

URL: http://kns.cnki.net/kcms/detail/11.1809.S.20190101.1031.002.html