韓潔楠 張 澤, 劉曉麗 李 冉 上官小川, 周婷芳, 潘 越 郝轉(zhuǎn)芳 翁建峰 雍洪軍 周志強 徐晶宇 李新海, 李明順,*

突變引起糯玉米籽粒淀粉積累差異研究

韓潔楠1張 澤1,2劉曉麗1李 冉1上官小川1,2周婷芳1,2潘 越1郝轉(zhuǎn)芳1翁建峰1雍洪軍1周志強1徐晶宇2李新海1,2李明順1,*

1中國農(nóng)業(yè)科學(xué)院作物科學(xué)研究所, 北京 100081;2黑龍江八一農(nóng)墾大學(xué)農(nóng)學(xué)院, 黑龍江大慶 163319

糯玉米是主要鮮食玉米類型,()基因?qū)肟稍黾幼蚜Ｙ嚢彼岷? 但同時引起籽粒皺縮、淀粉含量下降等, 限制了其育種應(yīng)用。為發(fā)掘優(yōu)良糯玉米受體, 以籽粒飽滿圓型近等基因系(-NIL)糯2/和皺縮型黃糯2/為研究材料, 通過對鮮食期、成熟期的百粒重和籽粒成分測定, 發(fā)現(xiàn)淀粉和可溶性糖含量不同可能是導(dǎo)致2份糯玉米-NILs表型差異的主要原因。利用實時熒光定量PCR技術(shù)分析, 發(fā)現(xiàn)授粉后10～24 d兩糯玉米-NILs中6個淀粉合成基因動態(tài)表達模式不同, 其中、、和差異較大。分析胚乳轉(zhuǎn)錄組數(shù)據(jù), 發(fā)現(xiàn)兩糯玉米-NILs中24個海藻糖和糖基水解酶編碼基因和48個胚乳修飾基因變化不同, 以上結(jié)果表明淀粉合成關(guān)鍵基因前期表達量高, 后期與對照無差異, 且糖代謝基因表達變化有利于淀粉合成可能是糯2/淀粉含量和百粒重不受突變影響, 籽粒性狀明顯優(yōu)于黃糯2/的重要原因, 同時多個胚乳修飾基因的差異表達可能與該結(jié)果直接相關(guān)。本研究結(jié)果可為突變體在玉米育種中的應(yīng)用提供重要參考。

糯玉米; 糯2/; 黃糯2/; 籽粒飽滿度; 淀粉; 糖代謝; 差異基因

玉米是全球種植面積最大的農(nóng)作物, 具有產(chǎn)量高、增產(chǎn)潛力大、用途廣等特點, 是人類食物、動物飼料及工業(yè)原料的重要來源[1]。玉米籽粒賴氨酸匱乏, 是制約營養(yǎng)價值的第一限制性氨基酸, 因此以玉米為主糧或飼用需額外添加賴氨酸。()為調(diào)控籽粒發(fā)育的核心轉(zhuǎn)錄因子, 其隱性突變體胚乳中賴氨酸含量比普通玉米提高約70%[2]。但突變導(dǎo)致胚乳變粉質(zhì)、容重及產(chǎn)量低、易感病。研究發(fā)現(xiàn)聚合胚乳修飾基因可使突變體籽?；謴?fù)為硬質(zhì)、產(chǎn)量提高, 從而選育出優(yōu)質(zhì)蛋白玉米(quality protein maize, QPM)[3-4]。QPM有效改善了南美、非洲和亞洲等以玉米為主食的發(fā)展中國家人群營養(yǎng)不良癥, QPM飼料也提高了畜禽蛋白利用率[5]。將突變基因?qū)肱从衩卓膳嘤齼?yōu)質(zhì)蛋白糯玉米, 但與連鎖的不良性狀導(dǎo)致糯玉米商品性差, 與普通QPM一樣優(yōu)質(zhì)蛋白糯玉米選育也需要積累胚乳修飾基因。

糯玉米起源于我國, 為9號染色體()基因突變引起。我國糯玉米品種資源豐富, 廣泛分布在云南、廣西一帶[6-7]。糯玉米適口性好、易消化、營養(yǎng)成分高于普通玉米, 是鮮食玉米的主力軍[8]。糯玉米若作青穗鮮食, 對果穗食用品質(zhì)和營養(yǎng)品質(zhì)有嚴格的要求。食用品質(zhì)主要是風(fēng)味、柔嫩性、含糖量、糯性及皮渣率等; 營養(yǎng)品質(zhì)主要包括蛋白質(zhì)含量、氨基酸組成、淀粉含量等[9-10]。分析我國40份溫帶糯玉米自交系賴氨酸含量為0.14%～0.39%, 平均含量僅為0.23%[11], 與人體及畜禽需求的0.5%差異較大[12]。為獲得兼具糯玉米和優(yōu)質(zhì)蛋白玉米優(yōu)良特性的糯玉米品種, 研究人員將基因?qū)肱从衩鬃越幌?。Misra等[13]發(fā)現(xiàn)雙突變體賴氨酸及游離氨基酸含量顯著提高。張述寬等[14]選育出18個高賴氨酸糯玉米自交系, 氨基酸含量高于QPM, 具有較好的抗病性和農(nóng)藝性狀。Sinkangam等[15]通過回交獲得11個高賴氨酸糯玉米, 賴氨酸、可溶性糖和支鏈淀粉含量顯著高于普通糯玉米。Zhou等[16]發(fā)現(xiàn)雙突變體醇溶蛋白、氨基酸合成、脅迫和信號轉(zhuǎn)導(dǎo)等相關(guān)蛋白質(zhì)積累發(fā)生變化, 支鏈淀粉含量增加。Dang等[17]運用雙單倍體技術(shù)發(fā)現(xiàn)聚合和基因可提高賴氨酸含量, 同時對糯玉米品質(zhì)影響較小。

對多個突變體進行轉(zhuǎn)錄組分析發(fā)現(xiàn), 一些基因僅在特定突變體/背景中表達水平有差異; 不同突變體中一致存在的差異表達基因, 表達水平因背景差異而明顯不同[18]。玉米種內(nèi)變異豐富, 因此突變引起的胚乳基因轉(zhuǎn)錄水平表達差異較大[19-23]。Jia等[20]發(fā)現(xiàn)W64A/中多個淀粉合成基因轉(zhuǎn)錄及蛋白水平上調(diào)表達, 認為、上調(diào)表達引起淀粉結(jié)晶度改變; Zhang等[22]發(fā)現(xiàn)W64A/中淀粉合成基因下調(diào)表達, 蛋白表達減少; 本課題組發(fā)現(xiàn)CAL58/中下調(diào)表達,、上調(diào)表達, SuSy酶活性降低, AGPase酶活性增加(數(shù)據(jù)未發(fā)表)。通過分子輔助育種可聚合和基因, 提高籽粒賴氨酸含量, 但雙突自交系籽粒容重低、易感病、產(chǎn)量下降, 不適宜直接選育品種[24-25]。突變體胚乳修飾基因數(shù)量多, 多數(shù)分子機制不清楚, 缺乏相應(yīng)的分子標記, 因此將及其修飾基因同時導(dǎo)入糯玉米自交系困難較大。通過比較多份糯玉米近等基因系(-NILs), 我們發(fā)現(xiàn)籽粒表型明顯不同[16,26], 表明存在優(yōu)良糯玉米受體, 在導(dǎo)入基因后籽粒產(chǎn)量與相應(yīng)普通糯玉米產(chǎn)量接近。本研究以籽粒表型顯著不同的-NILs黃糯2/和糯2/為材料, 通過比較鮮食期和成熟期籽粒組成、淀粉合成基因表達和轉(zhuǎn)錄組差異基因等, 為發(fā)掘優(yōu)良糯玉米受體親本提供依據(jù)。

1 材料與方法

1.1 田間種植和取樣

2020年5月初將試驗材料種植于中國農(nóng)業(yè)科學(xué)院作物科學(xué)研究所北京昌平試驗基地, 3 m行長, 25 cm株距, 每材料設(shè)置3個小區(qū), 每小區(qū)種植5行, 自交授粉并記錄授粉日期。用于轉(zhuǎn)錄組測序及定量分析的胚乳樣品通過人工剝?nèi)? 每個材料取3個重復(fù), 每重復(fù)取3個果穗置于冰上保鮮, 取果穗中部的籽粒, 在冰上用鑷子剝?nèi)∨呷椴⒀杆俜胚M液氮中冷凍暫存, 后置于–80℃冰箱中備用。授粉后25 d收獲鮮食期果穗, 每小區(qū)隨機選取3個穗, 混作一個生物學(xué)重復(fù), 部分新鮮籽粒保存?zhèn)溆? 另一部分籽粒在自然條件下晾干、脫粒, 去除雜損粒后裝袋保存?zhèn)溆?。其余果穗授粉后自然成熟并晾? 分小區(qū)收獲并脫粒后, 裝袋保存。

1.2 10K液相芯片技術(shù)檢測背景回復(fù)率

檢測樣品為回交6代自交3代以上的糯玉米回交轉(zhuǎn)育植株, 取田間種植下植株新葉樣品送石家莊博奧迪有限公司進行分析。在黃糯2一組材料中共檢測到11,444個核心SNP位點, 其中相同位點有11,359個, 差異位點有85個, 背景恢復(fù)率為99.26%; 糯2一組材料中共檢測到11,420個核心SNP, 差異位點有313個, 相同位點有11,107個, 背景恢復(fù)率為97.26%, 表明兩者與理論值98.4%接近。

1.3 百粒重、種皮厚度、可溶性糖、氨基酸及淀粉含量測定

百粒重用萬分之一天平稱量, 每小區(qū)隨機稱量3次100粒種子重量, 取平均值記作一個重復(fù)。鮮食期籽粒種皮厚度測定: 取授粉后25 d飽滿一致籽粒用FAA固定, 剝?nèi)∽蚜Ｅ弑趁婀? 在水和甘油混合液中軟化, 后置于27℃干燥, 用螺旋測微儀對每塊果皮測量3次, 取10粒果皮平均值記作一個重復(fù)。成熟期籽粒種皮厚度測定: 取飽滿一致籽粒10?；旌? 于沸水中煮, 冷卻后將背胚面種皮剝下, 吸水紙擦干后, 用螺旋測微尺對每塊種皮測量3次, 取平均值記作一個重復(fù), 具體測定方法參見劉曉麗等[27]。

賴氨酸測定依據(jù)《植物中游離氨基酸的測定》(GB/T 30987-2020)中全自動氨基酸分析儀法, 每份樣品測2次, 取平均值記作一個重復(fù), 委托青島科創(chuàng)質(zhì)量檢測有限公司進行。可溶性糖和總淀粉測定分別根據(jù)植物可溶性糖試劑盒(蒽酮比色法)以及Megazyme淀粉總量檢測試劑盒(K-TSTA)說明書操作, 每份樣品測2次, 取平均值記作一個重復(fù)。

1.4 醇溶蛋白的提取和SDS-PAGE電泳

將供試材料成熟籽粒分別研磨成粉, 稱取0.05 g置于離心管中, 加入70%乙醇400 μL (含2%巰基乙醇), 室溫搖晃2～3 h, 12,000轉(zhuǎn)min–1離心10 min, 吸取上清100 μL, 加入10% SDS溶液10 μL, 80℃烘干, 加入100 μL ddH2O重懸并加入Loading buffer煮沸, 吸取5 μL樣品進行10% SDS-PAGE凝膠電泳, 最后用固定液、染色液和脫色液處理直至條帶清晰可見, 詳細步驟參見劉曉麗等[27]。

1.5 轉(zhuǎn)錄組測序

對糯2/、黃糯2/及其輪回親本每個材料設(shè)置3次生物學(xué)重復(fù), 共計12個樣品進行轉(zhuǎn)錄組測序, 獲得90.73 GB數(shù)據(jù)量。各樣品Clean Data均達到6.42 GB, Q30堿基百分比在92.93%以上。將各樣品Clean Reads與B73參考基因進行序列比對, 比對率為86.05%～90.17%, 表明轉(zhuǎn)錄組測序的質(zhì)量較高。對12個樣品做系統(tǒng)進化樹分析, 糯2/與糯2、黃糯2/與黃糯2聚類在2個分支上, 表明所用材料和轉(zhuǎn)錄組數(shù)據(jù)具有較高可靠性。

1.6 RNA提取及cDNA合成

使用TransZolUP Plus RNA提取試劑盒(北京全式金生物技術(shù)股份有限公司)提取玉米胚乳RNA, 詳細操作步驟見說明書。使用FastKing gDNA Dispelling RT SuperMix試劑盒(天根生化科技(北京)有限公司)將RNA反轉(zhuǎn)為cDNA, 詳細操作步驟見說明書。得到的cDNA保存于–20℃冰箱。

1.7 熒光定量引物設(shè)計及定量驗證

以Ubiquitin為內(nèi)參基因, 對淀粉合成及糖代謝差異表達基因進行定量分析, 根據(jù)基因ID, 在NCBI數(shù)據(jù)庫中下載基因序列。將基因序列導(dǎo)入Primer 5.0設(shè)計定量引物, 委托北京華大基因科技有限公司合成。將cDNA稀釋作為模板, 通過普通PCR擴增目的條帶, 得到的PCR產(chǎn)物進行瓊脂糖凝膠電泳, 在紫外線照膠儀中觀察條帶是否特異, 本文所用定量引物信息見附表1。

采用cDNA模板2 μL, 10 μL 2×SuperReal PreMix Plus, 0.6 μL引物, 6.8 μL RNase-free ddH2O配制qRT-PCR反應(yīng)體系。每份樣品均設(shè)置3個生物學(xué)重復(fù)和3個技術(shù)重復(fù)。使用BIO-IQ5熒光定量PCR儀進行三步法PCR反應(yīng), 程序如下: 預(yù)變性95℃ 15 min,變性95℃ 10 s, 退火60℃ 10 s, 延伸72℃ 10 s, 40個循環(huán)。數(shù)據(jù)處理采用相對定量的分析方法采用2–ΔΔCT法, 計算公式為: 表達量比值=2–{[待測組目的基因平均CT值–待測組內(nèi)參基因平均CT值]–[對照組目的基因平均CT值–對照組內(nèi)參基因平均CT值]}

1.8 數(shù)據(jù)分析

數(shù)據(jù)通過Microsoft Excel 2007和SPSS 21.0進行統(tǒng)計分析。

2 結(jié)果與分析

2.1 糯玉米o2-NILs籽粒表型分析

前期我們以優(yōu)質(zhì)蛋白玉米CA339為基因供體, 通過回交轉(zhuǎn)育創(chuàng)制出多份糯玉米-NILs[16,25]。通過比較發(fā)現(xiàn)黃糯2/與糯2/籽粒表型顯著不同, 黃糯2/為典型的突變體表型, 籽粒明顯皺縮, 而糯2/籽粒呈飽滿圓潤狀, 皺縮不明顯, 但兩者胚乳均呈粉質(zhì)態(tài)(圖1-A)。與各自輪回親本相比(對照), 黃糯2/百粒重和淀粉含量顯著降低, 糯2/籽粒則無差異, 明顯高于黃糯2/并且該差異不受環(huán)境影響(圖1-B)。進一步測定籽粒成分, 鮮食期與對照相比黃糯2/總淀粉含量和百粒重降低6.12%和1.11%, 可溶性糖含量不變, 賴氨酸含量增加54.28%; 而糯2/籽粒總淀粉含量增加6.12%, 賴氨酸增加38.10%, 百粒重和可溶性糖含量不受影響。這與我們前期發(fā)現(xiàn)的突變對多份糯玉米鮮食期品質(zhì)影響較大, 黃糯2/和糯2/為代表性差異材料的結(jié)果一致[27]。成熟期黃糯2/籽粒中可溶性糖增加12.50%、總淀粉含量降低6.24%, 百粒重降低8.80%, 賴氨酸含量增加79.17%; 但糯2/籽?？偟矸酆?、可溶性糖及百粒重均不受影響, 賴氨酸含量增加58.33% (表1)。糯2/籽粒表型與對照相似, 但粉質(zhì)胚乳占比明顯增加, 因此胚乳質(zhì)地不同于QPM。黃糯2/胚乳中醇溶蛋白亞基27 kD γ-zein顯著降低, 糯2/胚乳中27 kD γ-zein含量顯著高于對照, 與CA339接近(圖2)。此外, 15 kD β-zein、19 kD和22 kD α-zein含量在糯玉米-NILs均明顯降低, 16 kD γ-zein在黃糯2/和糯2/變化相反。前期我們發(fā)現(xiàn)趙OP-6/SY1-2/與糯2/相似, 27 kD γ-zein明顯高于對照, 趙OP-6/淀粉含量增加, 籽粒較飽滿、圓潤而SY1-2/淀粉含量降低, 籽粒呈明顯皺縮狀[27], 表明27 kD γ-zein蛋白增加是糯玉米-NILs籽?；謴?fù)飽滿狀的必要非充分條件, 淀粉含量也是影響籽粒飽滿度的重要因素。

圖1 黃糯2/wx1wx1o2o2和糯2/wx1wx1o2o2籽粒表型

(A): 籽粒表型比較; (B): 不同年代下籽粒淀粉含量和百粒重比較。**表示在0.01概率水平差異顯著。

(A): kernel phenotype; (B): starch content and 100-kernel weight in different years. ** indicates significant difference at the 0.01 probability level.

2.2 糯玉米o2-NILs淀粉合成關(guān)鍵基因的動態(tài)表達分析

淀粉合成積累是一個持續(xù)過程, 顯著影響籽粒飽滿度。取授粉后10 d (10 DAP)、15 DAP、20 DAP、24 DAP胚乳對淀粉合成基因表達水平進行比較(圖3)。與各自對照相比, 10 DAP時兩糯玉米NILs中、、、和表達量均無顯著變化;和在黃糯2/中上調(diào)1.70倍和1.55倍, 在糯2/中上調(diào)1.99倍和3.22倍;在黃糯2/中上調(diào)1.52倍, 在糯2/中無顯著變化。15 DAP時,和在兩糯玉米NILs中無顯著變化;和在黃糯2/中下調(diào)1.69倍和2.22倍, 但在糯2/中無顯著變化;、、和在黃糯2/中無顯著變化, 在糯2/中則分別是對照的5.79、8.88、3.63和4.27倍。20 DAP時, 與對照相比黃糯2/中、、和無變化,和下調(diào)1.72倍和1.30倍,上調(diào)1.95倍和1.85倍; 糯2/中僅表達量下調(diào)3.13倍, 剩余基因均無變化。24 DAP時, 相較于對照黃糯2/中表達量下調(diào)1.37倍,、和上調(diào)1.48、1.45和1.54倍,、、和表達量無顯著變化; 而糯2/中所有供試基因無變化。綜上表明兩糯玉米NILs在授粉后15～24 d淀粉合成基因表達差異顯著, 其中、、和在3個時期發(fā)生顯著變化, 差異最明顯, 推測是導(dǎo)致兩糯玉米NILs籽粒淀粉含量不同的主要原因。

表1 鮮食期和成熟期糯玉米o2-NILs籽粒成分測定結(jié)果

*和**分別表示在0.05和0.01概率水平差異顯著。

*and**indicate significant difference at the 0.05 and 0.01 probability levels, respectively.

圖2 糯玉米胚乳醇溶蛋白亞基分析

2.3 糯玉米o2-NILs胚乳糖代謝路徑差異表達基因分析

為進一步探究突變導(dǎo)致黃糯2/和糯2/籽粒淀粉積累不同原因, 對兩材料20 DAP胚乳進行轉(zhuǎn)錄組測序。以各自輪回親本為對照分析黃糯2/和糯2/差異表達基因(differently expressed genes, DEGs), 黃糯2/中共有2466個DEGs, 其中1290個基因上調(diào)表達, 1176個基因下調(diào)表達; 糯2/中共有1685個DEGs, 其中1124個基因上調(diào)表達, 561個基因下調(diào)表達(圖4-A)。糯2/中DEGs遠低于黃糯2/, 表明突變對胚乳發(fā)育的影響依賴于基因型, 糯2/受影響程度可能低于黃糯2/。對DEGs進行KEGG路徑富集, 黃糯2/DEGs主要富集到內(nèi)質(zhì)網(wǎng)蛋白質(zhì)加工、碳代謝、DNA復(fù)制、淀粉和蔗糖代謝路徑(圖4-B); 糯2/主要富集到碳代謝、淀粉和蔗糖代謝、甘氨酸、絲氨酸和蘇氨酸代謝、苯丙烷生物合成和糖酵解5個路徑(圖4-C)。

授粉后20 d兩糯玉米-NILs共篩選出34個淀粉和糖代謝相關(guān)DEGs (圖5-A), 淀粉合成相關(guān)DEGs有AGPase小亞基、淀粉合成酶SSIII、葡萄糖變位酶、異淀粉酶和α淀粉酶編碼基因共計7個。剩余27個DEGs為糖代謝相關(guān)基因(圖5-B), 其中4個DEGs為兩糯玉米-NILs共有基因, 分別編碼丙酮酸磷酸雙激酶(PPDK)、海藻糖磷酸合酶(TPP)和果膠酯酶, 在兩糯玉米-NILs中一致下調(diào)。15個DEGs僅在黃糯2/中差異表達, 其中12個基因下調(diào), 3個基因上調(diào), 包括5個海藻糖六磷酸合酶(TPS)基因、3個TPP基因、4個糖基水解酶基因、2個果糖激酶、1個果膠酯酶; 7個DEGs在糯2/中特異表達, 其中5個基因下調(diào)表達, 2個基因上調(diào), 包括2個TPP編碼基因和5個糖基水解酶基因。對8個DEGs進行RT-PCR驗證, 發(fā)現(xiàn)與RNA-seq結(jié)果一致(圖6)。

圖3 糯玉米o2-NILs淀粉合成路徑關(guān)鍵基因差異表達量分析

*和**分別表示在0.05和0.01概率水平差異顯著。DAP: 授粉后天數(shù)。

* and ** indicate significant difference at the 0.05 and 0.01 probability levels, respectively.DAP: days after pollination.

圖4 糯玉米o2-NILs差異表達基因KEGG富集分析

(A): 兩糯玉米-NILs差異表達基因數(shù)目; (B): 黃糯2/差異表達基因KEGG富集分析; (C): 糯2/差異表達基因KEGG富集分析。

(A): the number of DEGs of the two waxy maize-NILs; (B): KEGG enrichment of differentially expressed genes in Huangnuo 2/; (C): KEGG enrichment of differentially expressed genes in Nuo 2/

圖5 授粉后20 d糯玉米o2-NILs淀粉和糖代謝路徑差異基因分析

(A): 糯玉米-NILs淀粉和糖代謝相關(guān)差異基因分析; (B): 糯玉米-NILs糖代謝差異基因分析。ns代表無顯著性差異。

(A): the analysis of differentially expressed genes in starch and sugar metabolism pathways of waxy maize-NILs; (B): the analysis of differentially expressed genes in sugar metabolic pathways of waxy maize-NILs. ns: no significant difference.

圖6 糯玉米o2-NILs糖代謝差異基因定量分析

*和**分別表示在0.05和0.01概率水平差異顯著。DAP: 授粉后天數(shù)。

* and ** indicate significant difference at the 0.05 and 0.01 probability levels, respectively.DAP: days after pollination.

糖代謝相關(guān)DEGs以海藻糖合成和糖基水解酶基因為主, 暗示糖代謝在兩糯玉米-NILs中發(fā)生顯著變化。動態(tài)表達分析黃糯2/中TPS編碼基因在10 DAP時表達量無變化, 但此后上調(diào)表達, 分別上調(diào)1.37、2.71和1.75倍, 糯2/中10 DAP時其表達量下調(diào)1.32倍, 15 DAP上調(diào)3.38倍, 20 DAP和24 DAP時無差異。TPP編碼基因在黃糯2/中10 DAP時表達量無變化, 15 DAP時上調(diào)4.46倍, 24 DAP時上調(diào)1.85倍, 在糯2/中10 DAP時表達量下調(diào)2.56倍, 20 DAP和24 DAP上調(diào)2.04倍和2.48倍; 另一TPP編碼基因在黃糯2/中顯著下調(diào)(1.40～3.33倍), 在糯2/中10 DAP和15 DAP下調(diào)2.86倍和3.85倍, 后2個時期不受影響。編碼葡聚糖-內(nèi)-1,3-β葡糖苷酶, 黃糯2/中15 DAP和24 DAP時表達量下調(diào)2.63和1.64倍, 但在10 DAP和20 DAP不受影響, 在糯2/中4個時期均下調(diào)表達(2.04～3.03倍);編碼另一個糖基水解酶, 黃糯2/中15 DAP時表達量下調(diào)2.78倍, 其余3個時期表達量與對照無差異, 在糯2/中4個時期表達量均顯著下調(diào)(2.50～3.45倍)。

2.4 糯玉米o2-NILs胚乳醇溶蛋白和氨基酸相關(guān)差異表達基因分析

黃糯2/中30個醇溶蛋白相關(guān)基因下調(diào)2.31～99.30倍(log2FC = –1.21～ –6.63), 糯2/中有24個醇溶蛋白基因下調(diào)2.30～ 45.21倍(log2FC = –1.20～ –5.50) (附表2)。其中22個為兩糯玉米-NILs共有DEGs, 19個基因編碼19 kD α-zein和22 kD α-zein,編碼15 kD β-zein, 20個基因在兩糯玉米-NILs中均下調(diào)表達, 與-NILs籽粒19 kD、22 kD和15 kD α-zein顯著降低結(jié)果一致;編碼16 kD γ-zein, 僅在黃糯2/中表達量下降,與其16 kD α-zein降低一致(圖2)。、、和為賴氨酸降解基因(附表3), 其下調(diào)1.2～17.1倍(log2FC = –1.01 ～ –4.10)使賴氨酸降解受阻, 可能是糯玉米-NILs賴氨酸含量增加的主要原因[28](表1)。

3 討論

玉米突變后籽粒表現(xiàn)不同, 例如導(dǎo)入自交系B46籽粒變化顯著, 而導(dǎo)入M14籽?；緹o變化[18]。譚華等[29]將導(dǎo)入多個普通玉米自交系后發(fā)現(xiàn)-NILs賴氨酸含量大幅提高, 容重增加, 熱帶和亞熱帶種質(zhì)百粒重多數(shù)下降, 但在溫帶種質(zhì)中增加。籽粒皺縮、胚乳粉質(zhì)且不透光是突變體的典型表現(xiàn)。淀粉是籽粒主要貯藏物質(zhì), 占胚乳干重的70%, 淀粉積累不足被認為是籽粒皺縮、百粒重低的重要原因。淀粉的合成是一個復(fù)雜連續(xù)過程, 授粉后10 d籽粒中淀粉開始大量合成, 20 d左右達到最大速率[30-32], 這一過程受蔗糖合成酶(SuSy)、腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、淀粉合成酶(SSs)、淀粉分支酶(SBE)和去分支酶(DBE)等關(guān)鍵酶調(diào)控, 編碼基因表達量的改變對籽粒淀粉含量影響顯著[33-35]。本研究發(fā)現(xiàn)黃糯2/和糯2/籽粒皺縮程度與胚乳淀粉合成基因表達不同密切相關(guān)(圖1、圖4和表1)。SuSy是雙向催化酶, 主要功能是分解蔗糖, 為淀粉和蛋白質(zhì)合成提供底物[36]。玉米K0326Y/中突變, SuSy活性降低, 淀粉合成受抑制, 籽粒皺縮[37]。黃糯2/中表達水平顯著下降, 籽粒明顯皺縮, 而糯2/中表達不受影響, 籽粒飽滿程度與對照接近。是SuSy的另一個編碼基因, 表達量變化與相似, 黃糯2/中下調(diào)而糯2/中無變化, 表明黃糯2/胚乳中淀粉合成底物供應(yīng)可能受到抑制, 但糯2/中不受影響。AGPase是淀粉合成限速酶, 影響碳向淀粉途徑的分配[38], 胚乳中主要編碼基因是和。正常籽粒授粉后蔗糖比重逐漸降低, 淀粉比重增加, 收獲時以淀粉為主, 而突變導(dǎo)致籽粒中多聚糖鏈合成受抑制, 蔗糖含量增加, 淀粉積累速率受抑制, 籽粒變?yōu)榘櫩s狀[39]。10 DAP時糯2/和黃糯2/中和顯著上調(diào), 但15 DAP時僅糯2/中上調(diào)表達(圖3), 表明淀粉積累初期(10 DAP)糯玉米NILs中AGPase表達增加, 但只有糯2/保持較高水平至15 DAP。SSs通過催化ADPG葡萄糖基轉(zhuǎn)移到葡聚糖的非還原末端來延長支鏈淀粉的長度, 糯玉米由于()基因突變, 直鏈淀粉合成受抑制, 只能合成支鏈淀粉; SBE將α-1,4糖苷鍵切開, 將截短的葡聚糖鏈與C6羥基鏈接, 形成支鏈淀粉的分支[40]。與對照比, 兩糯玉米-NIL中、表達水平無差異, 推測胚乳中短葡聚糖鏈的合成不受影響; 糯2/中15 DAP時上調(diào)表達, 與和變化一致, 表明前期糯2/可能積累更多支鏈淀粉, 與鮮食期籽粒淀粉含量高于對照相符; 黃糯2/中也上調(diào)表達, 推測胚乳中淀粉合成底物不足為主要限制因素, 因此籽粒淀粉含量低于對照。根據(jù)淀粉合成基因表達模式推測, 前期(0～15 DAP)糯2/籽粒淀粉合成速率較高, 20 DAP時恢復(fù)至對照水平, 但黃糯2/自15 DAP淀粉合成持續(xù)低于對照水平。

淀粉的合成還與糖代謝密切相關(guān), 例如海藻糖合成、糖酵解等均可與淀粉合成底物或中間產(chǎn)物相互轉(zhuǎn)化。海藻糖-6-磷酸酶(TPS)催化UDPG和葡萄糖-6-磷酸(G6P)合成海藻糖-6-磷酸(T6P), 進而在海藻糖-6-磷酸磷酸酶(TPP)作用下合成海藻糖。G6P和UDPG是淀粉合成和碳代謝底物, T6P為G6P和UDPG庫容標志物, 在植物生長及碳利用中有重要作用[41]。豌豆缺乏T6P, 種子大小和淀粉含量受影響,表型與孟德爾研究的皺縮豌豆種子相似[42]; 過表達大腸桿菌基因可增加擬南芥葉片淀粉合成[43]; Hu等[44]發(fā)現(xiàn)增強TPS或降低TPP活性可增加玉米籽粒淀粉含量。T6P還與蔗糖非發(fā)酵蛋白激酶(SnRK1)互作雙向調(diào)控蔗糖和淀粉合成[45]。植物TPS和TPP酶由多個基因編碼[46], 20 DAP時糯2/中基因表達不受影響, 多個基因下調(diào), 推測糯2/TPS活性不受影響,但TPP活性降低, 有利于淀粉合成; 而黃糯2/中多數(shù)和編碼基因下調(diào), 推測黃糯2/TPS和TPP活性均降低, 不利于淀粉生成(圖5-B)。進一步分析和動態(tài)表達模式說明海藻糖途徑少數(shù)基因的變化與淀粉合成速率直接相關(guān)(圖6)。果糖激酶在糖酵解途徑發(fā)揮重要作用, 可與蔗糖合酶協(xié)同調(diào)控蔗糖合成與降解[47], 番茄幼果中通過調(diào)節(jié)蔗糖輸入, 對淀粉積累產(chǎn)生影響[48-49]。本研究發(fā)現(xiàn)突變僅引起2個果糖激酶基因在黃糯2中下調(diào)表達, 是兩糯玉米-NILs糖酵解受影響不同的表現(xiàn)。結(jié)構(gòu)多樣的糖苷水解酶在糖和糖綴合物水解與合成中扮演重要角色, 編碼基因眾多, 例如擬南芥有393個編碼基因[50]。α-葡萄糖苷酶參與淀粉及糖原代謝, β-木糖酶與β-葡萄糖苷酶為纖維素酶, 降解纖維素生成纖維二糖和葡萄糖[51], 以上編碼基因在兩糯玉米-NILs多數(shù)下調(diào), 說明突變可能抑制了胚乳的糖苷水解, 使胚乳發(fā)育和細胞壁代謝受影響。果膠是大分子多糖, 與植物生長發(fā)育、逆境應(yīng)答等生物學(xué)過程密切聯(lián)系[52], 番茄中催化果膠多聚體形成的半乳糖醛酸轉(zhuǎn)移酶編碼基因突變, 果實果膠結(jié)構(gòu)改變, 淀粉和果實含量降低[53]。果膠酯酶是三大果膠酶之一, 隨機切除水溶性果膠分子的酯鍵, 產(chǎn)生游離羧基[54], 蘋果果膠甲酯酶在粉質(zhì)化不同的果實中呈現(xiàn)表達差異[55]。突變導(dǎo)致果膠酯酶基因下調(diào)表達, 可能與糯玉米-NILs胚乳質(zhì)地改變相關(guān)。磷酸丙酮酸雙激酶(PPDK)是CO2固定關(guān)鍵酶, 胚乳中高表達可與淀粉合成酶穩(wěn)定結(jié)合, 協(xié)同調(diào)控后期籽粒灌漿[56], 兩糯玉米NILs中一致下調(diào), 可能導(dǎo)致淀粉酶活性降低。

醇溶蛋白形成后儲存于蛋白體中, 若醇溶蛋白與淀粉同步合成, 則緊密結(jié)合形成硬質(zhì)胚乳占比高的硬粒玉米。糯玉米-NILs醇溶蛋白合成受阻, 淀粉合成基因也受影響(附表2和圖3), 導(dǎo)致成熟籽粒中儲藏物質(zhì)降低, 胚乳幾乎全部變?yōu)榉圪|(zhì)狀(圖1)。27-kD γ-zein調(diào)控蛋白體形成數(shù)量, QPM胚乳中γ27蛋白表達量增加, 是公認的修飾基因, 并且修飾基因數(shù)量與γ27蛋白表達正相關(guān)[57-58], 糯2/中27 kD γ-zein表達量高于黃糯2/(圖2), 暗示糯2/中胚乳修飾基因數(shù)量多。通過定位群體Holding發(fā)掘到多個胚乳修飾基因位點, 主效基因位于7號染色體; 包括27-kD γ-zein、葡萄糖轉(zhuǎn)運蛋白、焦磷酸依賴型果糖-6-磷酸1-磷酸轉(zhuǎn)移酶α亞基()、蛋白質(zhì)磷酸酶2C ()等16個基因位于連鎖區(qū)間內(nèi), 這些基因在QPM中多數(shù)上調(diào)表達[59-60]。Li等[61]發(fā)現(xiàn)熱激蛋白()、HSP伴侶蛋白()、絲氨酸/精氨酸富含蛋白質(zhì)編碼基因()在QPM中也呈上調(diào)表達。本研究發(fā)現(xiàn)黃糯2/中39個胚乳修飾基因差異表達, 包括32個、5個、1個和1個其中僅4個基因上調(diào); 糯2/中13個胚乳修飾基因差異表達, 包括6個、6個和1個, 其中8個基因上調(diào)(附表4)。表達增加使PFP催化活性提高, 加速糖酵解, 改善突變引起的能量匱乏, 與硬質(zhì)透明胚乳恢復(fù)相關(guān)[59-62];參與RNA剪切, 擬南芥中受脅迫時其上調(diào)表達[63], 水稻中在內(nèi)質(zhì)網(wǎng)應(yīng)激下被激活, 可促進未折疊蛋白在液泡和內(nèi)質(zhì)網(wǎng)之間的傳遞[64]。上述基因在糯玉米-NILs均下調(diào)表達, 是籽粒表型不同于普通糯玉米的重要原因。功能與脫落酸信號途徑有關(guān), 在優(yōu)質(zhì)蛋白玉米K0326Y/中上調(diào), 可能與其他修飾基因共同起作用[57], 但在糯玉米-NILs變化趨勢不一致。在QPM中上調(diào), 對緩解突變引起未折疊單重組或蛋白質(zhì)聚集引起的應(yīng)激效應(yīng)有重要作用[61-62], 糯2/中僅1個下調(diào), 而黃糯2/中有30個, 這可能與糯2/籽粒淀粉和百粒重不受影響直接相關(guān),需進一步研究。

4 結(jié)論

將基因?qū)肱从衩卓娠@著提高籽粒賴氨酸含量, 改善營養(yǎng)價值, 但與基因連鎖的不良性狀限制了優(yōu)質(zhì)蛋白糯玉米選育。與皺縮型近等基因系黃糯2/相比, 糯2/玉米成熟期淀粉含量及百粒重明顯提高, 籽粒圓潤飽滿。研究發(fā)現(xiàn)糯2/與黃糯2/中以、、和為主的淀粉合成基因、以海藻糖和糖基水解酶為主的糖代謝基因表達模式明顯不同, 推測糯2/10～15 DAP淀粉合成速率高, 后與對照無差異, 但黃糯2/自15 DAP起淀粉合成速率持續(xù)低于對照, 且糯2/中糖代謝基因的變化更有利于淀粉合成。根據(jù)轉(zhuǎn)錄組差異表達基因分析結(jié)果, 認為胚乳修飾基因, 尤其是熱激蛋白編碼基因的差異表達可能與兩糯玉米-NILs籽粒淀粉和百粒重不同直接相關(guān)。

[1] Ellis R P, Cochrane M P, Dale M F B, Duffus C M, Lynn A, Morrison I M, Prentice R D M, Swanston J S, Tiller S A. Starch production and industrial use., 1998, 77: 289–311.

[2] Mertz E T, Bates L S, Nelson O E. Mutant gene that changes protein composition and increases lysine content of maize endosperm., 1964, 145: 279–280.

[3] Paez A V, Helm J L, Zuber M S. Lysine content of opaque2 maize kernels having different phenotypes., 1969, 9: 251–253.

[4] Gibbon B C, Larkins B A. Molecular genetic approaches to developing quality protein maize., 2005, 21: 227–233.

[5] 石德權(quán). 優(yōu)質(zhì)蛋白玉米. 北京: 中國農(nóng)業(yè)出版社, 1995. Shi D Q. High Quality Protein Maize. Beijing: China Agriculture Press, 1995 (in Chinese).

[6] 曾孟潛. 我國糯質(zhì)玉米的親緣關(guān)系. 作物品種資源, 1987, (3): 4. Zeng M Q. The affinities of glutinous maize in China., 1987, (3): 4 (in Chinese).

[7] Zheng H J, Wang H, Yang H, Wu J H, Shi B, Cai R, Xu Y B, Wu A Z, Luo L J. Genetic diversity and molecular evolution of Chinese waxy maize germplasm., 2013, 8: e66606.

[8] 趙久然, 盧柏山, 史亞興, 徐麗. 我國糯玉米育種及產(chǎn)業(yè)發(fā)展動態(tài). 玉米科學(xué), 2016, 24(4): 67–71. Zhao J R, Lu B S, Shi Y X, Xu L. Dynamics of breeding and industrial development of glutinous maize in China., 2016, 24(4): 67–71 (in Chinese with English abstract).

[9] Azanza F, Klein B P, Juvik J A. Sensory characterization of sweet maize lines differing in physical and chemical composition., 1996, 61: 253–257.

[10] Simla S, Lertrat K, Suriharn B. Carbohydrate characters of six vegetable waxy maize varieties as affected by harvest time and storage duration., 2010, 9: 463–470.

[11] 楊引福, 郭強, 陳婧, 鄭小亞, 藺崇明. 中國溫帶糯玉米自交系遺傳及品質(zhì)性狀分析. 西北農(nóng)業(yè)學(xué)報, 2009, 29: 2213–2220. Yang Y F, Guo Q, Chen J, Zheng X Y, Lin C M. Analysis of genetic quality traits in temperate glutinous maize inbred lines in China., 2009, 29: 2213–2220 (in Chinese with English abstract).

[12] Young V R, Scrimshaw N S. Significance of Dietary Protein Source in Human Nutrition: Animal and/or Plant Proteins? online edn. New York: Oxford Academic, 1998. pp 205–221.

[13] Misra P S, Jambunathan R, Mertz E T, Glover D V, Barbosa H M, McWhirter K S. Endosperm protein synthesis in maize mutants with increased lysine content., 1972, 176: 1425–1427.

[14] 張述寬, 滕輝升, 蘇琪, 楊耀迥. 應(yīng)用SSR輔助選擇技術(shù)選育優(yōu)質(zhì)蛋白糯玉米自交系. 廣西農(nóng)業(yè)科學(xué), 2009, 40: 1279–1283. Zhang S K, Teng H S, Su Q, Yang Y J. Application of SSR-assisted selection technology to select high-quality protein glutinous maize inbred lines., 2009, 40: 1279–1283 (in Chinese with English abstract).

[15] Sinkangam B, Stamp P, Srinives P, Jompuk P, Mongkol W, Porniyom A, Dang N C, Jompuk C. Integration of quality protein in waxy maize by means of simple sequence repeat markers.,2011, 51: 2499–2504.

[16] Zhou Z Q, Song L Y, Zhang X X, Li X H, Yan N, Xia R P, Zhu H, Weng J F, Hao Z F, Zhang D G, Yong H J, Li M S, Zhang S H. Introgression of opaque2 into waxy maize causes extensive biochemical and proteomic changes in endosperm., 2016, 11: e0161924.

[17] Dang N C, Munsch M, Aulinger I, Renlai W, Stamp P. Inducer line generated double haploid seeds for combined waxy and opaque 2 grain quality in subtropical maize (L.)., 2012, 183: 153–160.

[18] Jia H W, Nettleton D, Peterson J M, Vazquez-Carrillo G, Jannink J L, Scott M P. Comparison of transcript profiles in wild-type and o2 maize endosperm in different genetic backgrounds., 2007, 47(S1): 45–59.

[19] Frizzi A, Caldo R A, Morrell J A, Wang M, Lutfiyya L L, Brown W E, Malvar T M, Huang S S. Compositional and transcriptional analyses of reduced zein kernels derived from themutation and RNAi suppression., 2010, 73: 569–585.

[20] Jia M, Wu H, Clay K L, Jung R, Larkins B A, Gibbon B C. Identification and characterization of lysine-rich proteins and starch biosynthesis genes in themutant by transcriptional and proteomic analysis., 2013, 13: 60.

[21] Li C B, Qiao Z Y, Qi W W, Wang Q, Yuan Y, Yang X, Tang Y P, Mei B, Lyu Y D, Zhao H, Xiao H, Song R. Genome-wide characterization of-acting DNA targets reveals the transcriptional regulatory framework ofin maize., 2015, 27: 532–545.

[22] Zhang Z Y, Zheng X X, Yang J, Messing J, Wu Y R. Maize endosperm-specific transcription factorsand PBF network the regulation of protein and starch synthesis., 2016, 113: 10842–10847.

[23] Zhan J P, Li G S, Ryu C-H, Ma C, Zhang S S, Lloyd A, Hunter B G, Larkins B A, Drews G N, Wang X F, Yadegari R.regulates a complex gene network associated with cell differentiation and storage functions of maize endosperm., 2018, 30: 2425–2446.

[24] 陳亮, 張德貴, 史振聲, 趙剛, 白麗, 張世煌, 李明順.突變基因()對玉米產(chǎn)量和產(chǎn)量配合力的影響. 玉米科學(xué), 2011, 19(1): 8–13. Chen L, Zhang D G, Shi Z S, Zhao G, Bai L, Zhang S H, Li M S. Effect ofmutant gene () on yield and yield fitness of maize., 2011, 19(1): 8–13 (in Chinese with English abstract).

[25] 宋麗雅, 陳亮, 何聰芬, 趙剛, 白鵬飛, 陳巖, 常馳.突變基因?qū)τ衩捉M合品質(zhì)的影響. 安徽農(nóng)業(yè)科學(xué), 2012, 40: 9607–9609. Song L Y, Chen L, He C F, Zhao G, Bai P F, Chen Y, Chang C. Effect ofmutant gene on the quality of maize combinations., 2012, 40: 9607–9609 (in Chinese with English abstract).

[26] 周昱婕, 韓潔楠, 王美娟, 劉曉麗, 李明順.基因?qū)ε从衩鬃恿Ｆ焚|(zhì)的影響分析. 玉米科學(xué), 2021, 29(2): 29–34. Zhou Y J, Han J N, Wang M J, Liu X L, Li M S. Analysis of the effect ofgene on kernel quality of glutinous maize., 2021, 29(2): 29–34 (in Chinese with English abstract).

[27] 劉曉麗, 韓潔楠, 李冉, 郭增輝, 張德貴, 李明順.對糯玉米籽粒食味和營養(yǎng)品質(zhì)的影響分析. 玉米科學(xué), 2023, 31(4): 52–58. Liu X L, Han J N, Li R, Guo Z H, Zhang D G, Li M S. Analysis of the effect ofon flavour and nutritional quality of glutinous maize kernels., 2023, 31(4): 52–58 (in Chinese with English abstract).

[28] Wang W, Dai Y, Wang M C, Yang W P, Zhao D G. Transcriptome dynamics of double recessive mutant,, reveals the transcriptional mechanisms in the increase of its lysine and tryptophan content in maize., 2019, 10: 316.

[29] 譚華, 鄒成林, 吳永升, 鄭德波, 莫潤秀, 黃愛花, 韋新興, 蔣維萍, 韋慧, 黃開健. 不同遺傳背景普通玉米種質(zhì)導(dǎo)入基因效應(yīng)探討. 廣東農(nóng)業(yè)科學(xué), 2015, 42(23): 127–132. Tan H, Zou C L, Wu Y S, Zheng D B, Mo R X, Huang A H, Wei X X, Jiang W P, Wei H, Huang K J. Exploration of the effect of introducinggene in common maize germplasm with different genetic backgrounds., 2015, 42(23): 127–132 (in Chinese with English abstract).

[30] Prioul J L, Mechin V, Lessard P, Thévenot C, Grimmer M, Chateau-Joubert S, Coates S, Hartings H, Kloiber-Maitz M, Murigneux A, Sarda X, Damerval C, Edwards K J. A joint transcriptomic, proteomic and metabolic analysis of maize endosperm development and starch filling., 2008, 6: 855–869.

[31] Chen J, Zeng B, Zhang M, Xie S J, Wang G K, Hauck A, Lai J S. Dynamic transcriptome landscape of maize embryo and endosperm development., 2014, 166: 252–264.

[32] Ji C, Xu L N, Li Y J, Fu Y X, Li S, Wang Q, Zeng X, Zhang Z Q, Zhang Z Y, Wang W Q, Wang J C, Wu Y R. The-transcriptional regulatory network orchestrates the coordination of endosperm cell expansion and grain filling in maize., 2022, 15: 468–487.

[33] Li N, Zhang S J, Zhao Y J, Li B, Zhang J R. Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize., 2011, 233: 241–250.

[34] Jiang L L, Yu X M, Qi X, Yu Q, Deng S, Bai B, Li N, Zhang A, Zhu C F, Liu B, Pang J S. Multigene engineering of starch biosynthesis in maize endosperm increases the total starch content and the proportion of amylose., 2013, 22: 1133–1142.

[35] Hu S T, Wang M, Zhang X, Chen W K, Song X R, Fu X Y, Fang H, Xu J, Xiao Y N, Li Y R, Bai G H, Li J S, Yang X H. Genetic basis of kernel starch content decoded in a maize multi-parent population., 2021, 19: 2192–2205.

[36] Cobb B G, Hannah L C. Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm., 1988, 88: 1219–1221.

[37] Deng Y T, Wang J C, Zhang Z Y, Wu Y R. Transactivation of Sus1 and Sus2 byis an essential supplement to sucrose synthase-mediated endosperm filling in maize., 2020, 18: 1897–1907.

[38] Denyer K, Dunlap F, Thorbj?rnsen T, Keeling P, Smith A M. The major form of ADP-glucose pyrophosphorylase in maize endosperm is extra-plastidial., 1996, 112: 779–785.

[39] Jennings P H, McCombs C L. Effects of sugary-1 and shrunken-2 loci on kernel carbohydrate contents, phosphorylase and branching enzyme activities during maize kernel ontogeny., 1969, 8: 1357–1363.

[40] Tetlow I J, Beisel K G, Cameron S, Makhmoudova A, Liu F, Bresolin N S, Wait R, Morell M K, Emes M J. Analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes., 2008, 146: 1878–1891.

[41] Paul M J, Watson A, Griffiths C A. Trehalose 6-phosphate signalling and impact on crop yield., 2020, 48: 2127–2137.

[42] Meitzel T, Radchuk R, McAdam E L, Thorm?hlen I, Feil R, Munz E, Hilo A, Geigenberger P, Ross J J, Lunn J E, Borisjuk L. Trehalose 6-phosphate promotes seed filling by activating auxin biosynthesis., 2021, 229: 1553–1565.

[43] Kolbe A, Tiessen A, Schluepmann H, Paul M, Ulrich S, Geigenberger P. Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase., 2005, 102: 11118–11123.

[44] Hu S T, Wang M, Zhang X, Chen W K, Song X R, Fu X Y, Fang H, Xu J, Xiao Y N, Li Y R, Bai G H, Li J S, Yang X H. Genetic basis of kernel starch content decoded in a maize multi-parent population., 2021, 19: 2192–2205.

[45] Fernandez O, Vandesteene L, Feil R, Baillieul F, Lunn J E, Clément C. Trehalose metabolism is activated upon chilling in grapevine and might participate ininduced chilling tolerance., 2012, 236: 355–369.

[46] Leyman B, Dijck P V, Thevelein J M. An unexpected plethora of trehalose biosynthesis genes in., 2001, 6: 510–513.

[47] Davies H V, Shepherd L V, Burrell M M, Carrari F, Urbanczyk-Wochniak E, Leisse A, Hancock R D, Taylor M, Viola R, Ross H, McRae D, Willmitzer L, Fernie A R. Modulation of fructokinase activity of potato () results in substantial shifts in tuber metabolism., 2005, 46: 1103–1115.

[48] Schaffer A A, Petreikov M. Sucrose-to-starch metabolism in tomato fruit undergoing transient starch accumulation., 1997, 113: 739–746.

[49] German M A, Dai N, Matsevitz T, Hanael R, Petreikov M, Bernstein N, Ioffe M, Shahak Y, Schaffer A A, Granot D. Suppression of fructokinase encoded by LeFRK2 in tomato stem inhibits growth and causes wilting of young leaves., 2003, 34: 837–846.

[50] Urbanowicz B R, Bennett A B, Del Campillo E, Catalá C, Hayashi T, Henrissat B, H?fte H, McQueen-Mason S J, Patterson S E, Shoseyov O, Teeri T T, Rose J K. Structural organization and a standardized nomenclature for plant endo-1,4-beta-glucanases (cellulases) of glycosyl hydrolase family 9., 2007, 144: 1693–1696.

[51] 潘利華, 羅建平. β-葡萄糖苷酶的研究及應(yīng)用進展. 食品科學(xué), 2006, 27: 803–807. Pan L H, Luo J P. Progress of research and application of β-glucosidase., 2006, 27: 803–807 (in Chinese with English abstract).

[52] 陳凱莉, 許軻, 張賢聰, 王亞楠, 汪志輝, 王迅. 果實中果膠代謝相關(guān)酶基因的研究進展. 園藝學(xué)報, 2017, 44: 2008–2014. Chen K L, Xu K, Zhang X C, Wang Y N, Wang Z H, Wang X. Progress of pectin metabolism-related enzyme genes in fruits., 2017, 44: 2008–2014 (in Chinese with English abstract).

[53] Godoy F D, Bermúdez L, Lira B S, Souza A P D, Elbl P, Dcmarco D, Alseekh S, Insani M, Buckeridge M, Almeida J, Grigioni G, FernieA R, Carrari F, Rossi M. Galacturonosyl transferase 4 silencing alters pectin composition and carbon partitioning in tomato., 2013, 64: 2449–2466.

[54] 傅海, 趙佳, 李偉, 孫科, 王希信. 果膠酶研究進展及應(yīng)用. 生物化工, 2020, 6(5): 148–153. Fu H, Zhao J, Li W, Sun K, Wang X X. Research progress and application of pectinase., 2020, 6(5): 148–153 (in Chinese with English abstract).

[55] Segonne S M, Bruneau M, Celton J M, Gall S L, Francin-Allami M, Juchaux M, Laurens F, Orsel M, Penou J P. Multiscale investigation of mealiness in apple: an atypical role for a pectin methylesterase during fruit maturation., 2014, 14: 375.

[56] Hennen-Bierwagen T A, Lin Q, Grimaud F, Planchot V, Keeling PL, James M G, Myers A M. Proteins from multiple metabolic pathways associate with starch biosynthetic enzymes in high molecular weight complexes: a model for regulation of carbon allocation in maize amyloplasts., 2009, 149: 1541–1559.

[57] Wang W, Niu S Z, Dai Y, Wang M C, Li Y, Yang W P, Zhao D G. Themutantsand opaque16 disclose lysine change in waxy maize as revealed by RNA-seq., 2019, 9: 12265.

[58] Lopes M A, Takasaki K, Bostwick D E, Helentjaris T, Larkins B A. Identification of two opaque2 modifier loci in quality protein maize., 1995, 247: 603–613.

[59] Holding D R, Hunter B G, Chung T, Gibbo B C, Ford C F, Bharti A K, Messing J, Hamaker B R, Larkins B A. Genetic analysis ofmodifier loci in quality protein maize., 2008, 117: 157–170.

[60] Holding D R, Hunter B G, Klingler J P, Wu S, Guo X M, Gibbon B C, Wu R L, Schulze J M, Jung R, Larkins B A. Characterization ofmodifier QTLs and candidate genes in recombinant inbred lines derived from the K0326Y quality protein maize inbred., 2011, 122: 783–794.

[61] Li C S, Xiang X L, Huang Y C, Zhou Y, An D, Dong J Q, Zhao C X, Liu H J, Li Y B, Wang Q, Du C G, Messing J, Larkins B A, Wu Y R, Wang W Q. Long-read sequencing reveals genomic structural variations that underlie creation of quality protein maize., 2020, 11: 17.

[62] Guo X M, Ronhovde K, Yuan L L, Yao B, Soundararajan M P, Elthon T, Zhang C, Holding D R. Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase induction and attenuation ofgene expression during endosperm modification in quality protein maize., 2012, 158: 917–929.

[63] Tanabe N, Yoshimura K, Kimura A, Yabuta Y, Shigeoka S. Differential expression of alternatively spliced mRNAs ofSR protein homologs, atSR30 and atSR45a, in response to environmental stress., 2007, 48: 1036–1049.

[64] Ohta M, Takaiwa F. Emerging features of ER resident J-proteins in plants., 2014, 9: e28194.

附表1 定量引物信息列表

Table S1 Information of RT-PCR primers

基因名稱Gene ID引物序列Primer sequence (5'–3')片段長度Production size (bp)退火溫度Annealing temperature (℃) Zm00001d045042(Sh1)TGTTTCACCGCAATTCGCA19060 AGACAGGTGAACGAGCAGGC Zm00001d029091(Sus1)GAAGCGTTCGGTCTCACC16260 GAAGAAGTCGGCCATCAG Zm00001d050032(Bt2)CTATGACCGTTTTGCTCCAAT19560 GCACCCATTAGTAAACTGTCCTC Zm00001d044129(Sh2)CGTGTCAGCTCTGGATGTGAA21060 AGCCTCTTGGATGCCCTTA Zm00001d045261(SSI)GACATTAATGATTGGAACCCTGCC18760 GAATGAGATCAATGCCTTTCTG Zm00001d037234(SSIIa)CTGCACTCCTGCCTGTTTAT17560 GGATCGTACAGCTCGAAATGTT Zm00001d000002(SSIIIa)CAAAAGGGGATCCACCTGATCA20860 CAGAGCCAGCGTATATCAGAT Zm00001d016684(SBEIIb)TAGACTTATCACAATGGGTTTAGGA22760 GCATTGCCTGATCAAACTCTTG Zm00001d017502GTCGCAGGACTCGTGAAACA20660 TTGAAGGCGTCTTCGTCTGT Zm00001d032311GTATGGCACTATTTGGACGCG18660 GACCAGTGTGTGAATCAGCTTG

(續(xù)附表1)

基因名稱Gene ID引物序列Primer sequence (5'–3')片段長度Production size (bp)退火溫度Annealing temperature (℃) Zm00001d052060TGAATCGTCTGTGCGAGGACC16460 CCACCTTGTGAAGTAACCGTGCT Zm00001d012748GGTTACTTCTTGAGGTGGTCT20860 GCATCTCCTTTGCCTGTGAG Zm00001d029371CCCGCCGAGGTCAAGGAGTT16060 TGCCGGTGCAGCAGGTAGGT Zm00001d012173ATCACCGCCACGAAGAAGGG21060 TAGGAACTAGGGCAAGCAAA Zm00001d042536GCCTTCCTTCAAAGTACAACA22060 CTCTATTAGCTGCAAGACCTCC Zm00001d036608ACCTCCGCTACTCCATCAACACCA23460 CCCTTGCGAACGGGTAGAACG Zm00001d034017TCTCCACCGTCATGATCTCCTA20060 AATGCCAGCAAGAATCGAAGCC Zm00001d033649GGAGAAGTATGGGAATCCAACG21360 TACCCTGCCAGCCACTCGAAG Zm00001d028243GCCTTCAACGCCTACTACCACG14460 GGTGCCGTTCATCAACGACGTC Zm00001d005546CAGCACGAGTGTTCTTGGGATC19960 GCGGTTGAGCGAAGCAGAGT Zm00001d053960GCTGGAGCTTTTGGTCAGTTTGC19060 GGTCGCACCAGATACTGAAATCT Zm00001d002256CGACAGAGCCATAACCACAT17260 AAACGAGCCTGATTTCCCTA Zm00001d015327(Ubiquitin)TAAGCTGCCGATGTGCCTGCGTCG20660 CTGAAAGACAGAACATAATGAGCACAG

附表2 糯玉米-NILs醇溶蛋白相關(guān)DEGs

Table S2 DEGs related to zein protein in waxy maize-NILs

基因名稱Gene ID糯2/wx1wx1o2o2Nuo 2/wx1wx1o2o2log2 FC黃糯2/wx1wx1o2o2Huangnuo 2/wx1wx1o2o2log2 FC注釋Annotation Zm00001d005793ns–1.21Prolamin 16 kD gamma zein precursor Zm00001d048847–1.80–5.05Prolamin 19 kD alpha zein z1A1_2 precursor Zm00001d048848ns–3.56Prolamin 19 kD alpha zein z1A1_3 precursor Zm00001d048849ns–3.63Prolamin 19 kD alpha zein z1A1_4 precursor Zm00001d048850–1.34–3.67Prolamin 19 kD alpha zein z1A1_5 precursor Zm00001d048851ns–3.90Prolamin 19 kD alpha zein z1A1_6 precursor Zm00001d048852ns–3.76Prolamin 19 kD alpha zein z1A1_7 precursor Zm00001d019155–1.36–3.70Prolamin 19 kD alpha zein z1B_4 precursor Zm00001d030855–1.33–2.18Prolamin 19 kD alpha zein z1D_4 precursor newGene_32946–1.67–2.30Prolamin 19 kD alpha zein z1D_2 precursor newGene_32956–2.52–2.73Prolamin 19 kD alpha zein z1D_2 precursor newGene_17461ns–3.84Prolamin 19 kD alpha zein z1A2_2 precursor newGene_33790–2.19–3.66Prolamin 19 kD alpha zein z1B_1 precursor

(續(xù)附表2)

基因名稱Gene ID糯2/wx1wx1o2o2Nuo 2/wx1wx1o2o2log2 FC黃糯2/wx1wx1o2o2Huangnuo 2/wx1wx1o2o2log2 FC注釋Annotation Zm00001d048816–3.78–4.24Prolamin 22 kD alpha zein z1C1_10 precursor Zm00001d048806–2.78–6.63Prolamin 22 kD alpha zein z1C1_12 precursor Zm00001d048817–5.20–5.80Prolamin 22 kD alpha zein z1C1_19 precursor Zm00001d048812–5.50–5.71Prolamin 22 kD alpha zein z1C1_7 precursor Zm00001d048813–2.75–3.58Prolamin 22 kD alpha zein z1C1_8 precursor Zm00001d049243–4.90–5.63Prolamin 22 kD alpha zein z1C2 precursor Zm00001d048810–3.69–4.77Prolamin 22 kD alpha-zein 14 Zm00001d048809-4.49–4.61Prolamin 22 kD alpha-zein 4 Zm00001d048818–4.61–4.30Prolamin 22 kD alpha-zein 8 Zm00001d020591ns–1.87Prolamin 50 kD gamma zein Zm00001d035760–1.70nsProlamin PPROL 17 precursor Zm00001d049476–1.36–2.90Z1A alpha zein protein Zm00001d045937ns–3.18Zein protein Zm00001d048807–4.37–4.79Zein seed storage protein, hypothetical protein Zm00001d019160–2.11–4.64Zein seed storage protein, hypothetical protein Zm00001d019162–1.49–5.11Zein seed storage protein, hypothetical protein Zm00001d019156–1.22–3.64Zein seed storage protein, hypothetical protein Zm00001d013100–1.20nsZein-binding Zm00001d048808–4.10–4.96Kafirin PSKR2 Precursor

附表3 糯玉米-NILs賴氨酸降解相關(guān)DEGs

Table S3 DEGs related to lysine degradation in waxy maize-NILs

基因名稱Gene_ID糯2/wx1wx1o2o2Nuo 2/wx1wx1o2o2log2 FC黃糯2/wx1wx1o2o2Huangnuo 2/wx1wx1o2o2log2 FC注釋Annotation Zm00001d020984–2.43–4.10Probable sarcosine oxidase Zm00001d003983–2.48nsAldehyde dehydrogenase family 7 member A1 Zm00001d008432ns–1.01Putative acetyl-CoA acetyltransferase cytosolic 2 Zm00001d052079ns–2.65Lysine-ketoglutarate reductase/saccharopine dehydrogenase1

附表4 糯玉米-NILs胚乳修飾相關(guān)DEGs

Table S4 DEGs related to endosperm modification in waxy maize-NILs

基因名稱Gene_ID糯2/wx1wx1o2o2Nuo 2/wx1wx1o2o2log2FC黃糯2/wx1wx1o2o2Huangnuo 2/wx1wx1o2o2log2FC注釋Annotation Zm00001d0155041.701.70Protein phosphatase 2C isoform gamma Zm00001d0399421.12–2.2816.9 kD class I heat shock protein 3 Zm00001d0377172.25–1.70Heat shock 70 kD protein 14 Zm00001d011241–1.58ns15.7 kD heat shock protein Zm00001d0395661.20ns17.5 kD class II heat shock protein Zm00001d0088411.61ns17.8 kD class II heat shock protein Zm00001d0313291.14nsCatalytic/protein phosphatase type 2C Zm00001d039933ns–1.0216.9 kD class I heat shock protein 1 Zm00001d039936ns–1.5416.9 kD class I heat shock protein 1 Zm00001d017813ns–2.4217.8 kD heat shock protein isoform X4

(續(xù)附表4)

基因名稱Gene_ID糯2/wx1wx1o2o2Nuo 2/wx1wx1o2o2log2FC黃糯2/wx1wx1o2o2Huangnuo 2/wx1wx1o2o2log2FC注釋Annotation Zm00001d028557ns–1.4717.9 kD class I heat shock protein Zm00001d028561ns–2.1117.9 kD class I heat shock protein Zm00001d047841ns–2.3217.9 kD class I heat shock protein Zm00001d028555ns–1.6418.1 kD class I heat shock protein Zm00001d003554ns–1.2422.0 kD class IV heat shock protein precursor Zm00001d025508ns–1.4622.0 kD class IV heat shock protein precursor Zm00001d010693ns1.2122.3 kD class VI heat shock protein Zm00001d050119ns–1.44Activator of 90 kDa heat shock protein ATPase Zm00001d015227ns–2.02Activator of Hsp90 ATPase Zm00001d031740ns–1.38Activator of Hsp90 ATPase Zm00001d015777ns–1.90Chloroplast small heat shock protein Zm00001d018298ns–1.59Class II heat shock protein Zm00001d039259ns–1.30DNAJ heat shock family protein Zm00001d047726ns–1.71DnaJ protein ERDJ3A Zea_mays_new Gene_4468ns–1.13Heat shock 70 kD protein Zm00001d048073ns–1.88Heat shock 70 kD protein 1 Zm00001d042922ns–1.17Heat shock 70 kD protein-like Zm00001d047799ns–1.81Heat shock cognate 70 kD protein Zm00001d028630ns–1.75Heat shock cognate 70 kD protein 2 Zm00001d012420ns–2.06Heat shock protein 1 Zm00001d024903ns–1.71Heat shock protein 82 Zm00001d020898ns–1.33Heat shock protein 90-2 Zm00001d031332ns–1.32Heat shock protein 90-2 Zm00001d039935ns–1.34Heat shock protein 17-2 Zm00001d028408ns–2.16Heat shock protein26 Zm00001d030346ns2.95Hsp20/alpha crystallin family protein Zm00001d002542ns1.32Probable protein phosphatase 2C Zea_mays_new Gene_6471.62nsProbable protein phosphatase 2C Zm00001d021817–1.01nsProbable protein phosphatase 2C Zm00001d017643–1.48nsProbable protein phosphatase 2C 25 Zm00001d047807–2.29nsprobable protein phosphatase 2C 31 Zm00001d0201001.23nsProbable protein phosphatase 2C 68 Zm00001d045919–1.88nsPyrophosphate-fructose 6-phosphate-Phosphotransferase subunit alpha 2 Zm00001d028615ns–1.39Probable protein phosphatase 2C 31 Zm00001d025055ns–1.88Probable protein phosphatase 2C 37 Zm00001d011195ns–1.35Probable protein phosphatase 2C 38 Zm00001d047847ns–1.19Serine/arginine-rich splicing factor SR45a Zm00001d031325ns–2.33Small heat shock protein Zm00001d032893ns–1.78Small heat shock-like protein

Analysis of differential accumulation of starch in waxy maize grain caused by themutation gene

HAN Jie-Nan1, ZHANG Ze1,2, LIU Xiao-Li1, LI Ran1, SHANG-GUAN Xiao-Chuan1,2, ZHOU Ting-Fang1,2, PAN Yue1, HAO Zhuan-Fang1, WENG Jian-Feng1, YONG Hong-Jun1, ZHOU Zhi-Qiang1, XU Jing-Yu2, LI Xin-Hai1,2, and LI Ming-Shun1,*

1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China;2College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang, China

The primary variety of fresh maize known as waxy maize undergoes a transformation with the introduction of the() mutant gene, resulting in an increased lysine content, thus improving the grain's nutritional composition. Yet, themutation brings about desirable agronomic traits such as wrinkle formation and a decrease in starch content, which restrict its use in breeding applications. To explore the high performing waxy maizereceptors, we capitalized on the use ofnear-isogenic line (-NIL), specifically the plump and round grain type Nuo 2/and its wrinkled counterpart, Huangnuo2/. Measurements of 100-grain weight and grain composition at the fresh ear and mature stages showed that there was difference in starch and soluble sugar content, which might be the primary cause of kernel phenotype variation between the two waxy maize-NILs. Genetic analysis of starch synthesis in the two-NILs was performed using qRT-PCR technique revealed that six gene-regulated trends fluctuated between 10 and 24 days after pollination, among which,,, andgenes were significant differences. Endosperm transcriptomes indicated that 24 genes encoding trehalose and glycosyl hydrolases and 48 genes involved in endosperm modification exhibited distinct changes between the two-NILs. There was no detectable alteration in the 100-grain weight or the starch content of Nuo 2/, which may well be tied to the early high-level expression of the primary starch synthesis gene, leaving later stages unchanged compared with the control. Furthermore, the shifts in the expression of sugar metabolism genes was beneficial to starch synthesis, which may be an important reason why starch content and 100-kernel weight of Nuo 2/were unaffected by themutation, and grain traits were significantly better than the superior grain traits compared with Huangnuo 2/. These results may be directly related to the differential expression of multiple endosperm modifying genes. The results of this study can provide important reference for the future utilization ofmutants in maize breeding.

waxy maize; Nuo 2/; Huangnuo 2/; kernel fullness degree; starch; sucrose metabolism; different expression genes

10.3724/SP.J.1006.2024.33046

本研究由國家重點研發(fā)計劃項目(2021YFD1201004)和財政部和農(nóng)業(yè)農(nóng)村部國家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(玉米, CARS-02)資助。

This study was supported by the National Key Research and Development Program of China (2021YFD1201004) and the China Agriculture Research System of MOF and MARA (Maize, CARS-02).

李明順, E-mail: limingshun@caas.cn

E-mail: hanjienan@caas.cn

2023-08-01;

2023-10-23;

2023-11-13.

URL: https://link.cnki.net/urlid/11.1809.S.20231110.0845.002

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