灌漿期夜間高溫脅迫下耐熱和熱敏感水稻籽粒的比較蛋白質(zhì)組分析

黃小平張宏玉雷剛王志美章智賀超廖江林,,?黃英金,,?

(1江西農(nóng)業(yè)大學(xué)作物生理生態(tài)與遺傳育種教育部重點(diǎn)實(shí)驗(yàn)室,南昌330045;2江西農(nóng)業(yè)大學(xué)農(nóng)業(yè)應(yīng)對(duì)氣候變化南昌市重點(diǎn)實(shí)驗(yàn)室,南昌330045;?通訊聯(lián)系人,E-mail:jlliao514815＠163．com,yjhuang_cn＠126．com)

灌漿期夜間高溫脅迫下耐熱和熱敏感水稻籽粒的比較蛋白質(zhì)組分析

黃小平1張宏玉2雷剛1王志美1章智1賀超1廖江林1,2,?黃英金1,2,?

(1江西農(nóng)業(yè)大學(xué)作物生理生態(tài)與遺傳育種教育部重點(diǎn)實(shí)驗(yàn)室,南昌330045;2江西農(nóng)業(yè)大學(xué)農(nóng)業(yè)應(yīng)對(duì)氣候變化南昌市重點(diǎn)實(shí)驗(yàn)室,南昌330045;?通訊聯(lián)系人,E-mail:jlliao514815＠163．com,yjhuang_cn＠126．com)

【目的】為了闡明水稻灌漿期耐夜間高溫的分子機(jī)制,提高水稻耐熱性,鑒定了水稻近等基因系耐熱純系XN0437T與熱敏感純系XN0437S在灌漿期夜間高溫脅迫下的差異表達(dá)蛋白質(zhì)?！痉椒ā坎捎猛霸苑椒ㄅ嘤?于開花期標(biāo)記同一天揚(yáng)花的穎花以保障所取穎花樣品的生育進(jìn)程一致;于水稻灌漿初期(花后第8天)進(jìn)行夜間高溫處理。高溫處理結(jié)束后剪取帶標(biāo)記的穎花提取水稻籽?？偟鞍踪|(zhì),采用8-plex i TRAQ試劑盒進(jìn)行蛋白質(zhì)樣品的差異顯色標(biāo)記,標(biāo)記樣品采用高效液相系統(tǒng)進(jìn)行質(zhì)譜鑒定及定量分析。【結(jié)果】鑒定并定量了3130個(gè)蛋白質(zhì),耐熱與熱敏感水稻純系間存在36個(gè)差異表達(dá)蛋白質(zhì)。蛋白質(zhì)功能注釋結(jié)果顯示,鑒定的36個(gè)差異表達(dá)蛋白質(zhì)中僅14個(gè)蛋白質(zhì)(占38．9%)注釋了功能,12個(gè)蛋白質(zhì)(占33．3%)為推測(cè)性功能注釋,10個(gè)(占27．8%)為功能未知的蛋白質(zhì)。已注釋功能的14個(gè)蛋白質(zhì)中,5個(gè)蛋白質(zhì)參與能量代謝,3個(gè)蛋白質(zhì)參與物質(zhì)轉(zhuǎn)運(yùn)與代謝,2個(gè)蛋白質(zhì)參與光合作用,3個(gè)蛋白質(zhì)為響應(yīng)逆境的鋅指蛋白質(zhì),1個(gè)蛋白質(zhì)為響應(yīng)逆境的熱激蛋白質(zhì)。【結(jié)論】灌漿期夜間高溫影響水稻籽粒細(xì)胞內(nèi)參與能量代謝、物質(zhì)轉(zhuǎn)運(yùn)與代謝、光合作用等相關(guān)蛋白質(zhì)的表達(dá)模式。水稻籽粒細(xì)胞中鋅指蛋白質(zhì)Q67 TK9、Q10N88上調(diào)表達(dá),鋅指蛋白質(zhì)Q5YLY5下調(diào)表達(dá),有利于提高水稻灌漿期對(duì)夜間高溫的耐熱性。

水稻;灌漿期;夜間高溫;蛋白質(zhì)組學(xué);iTRAQ技術(shù)

全球氣候變暖,尤其是夏季頻繁高溫對(duì)水稻等作物產(chǎn)量和品質(zhì)的不利影響,已受到廣泛關(guān)注[1,2]。在全球氣溫升高的大趨勢(shì)下,夜間氣溫的升高幅度顯著大于白天。而且夜間平均氣溫每升高1℃可導(dǎo)致水稻減產(chǎn)10%,而白天氣溫的升高與水稻產(chǎn)量間無(wú)顯著相關(guān)性[3],表明夜間高溫是直接導(dǎo)致水稻減產(chǎn)的主要原因[4]。在我國(guó),夏季高溫?zé)岷χ饕l(fā)生在長(zhǎng)江中下游地區(qū),尤其在7月份,該區(qū)域受西太平洋副熱帶高壓控制,天氣晴朗、太陽(yáng)輻射強(qiáng)烈,極易出現(xiàn)高溫酷熱天氣[5,6]。該區(qū)域是我國(guó)雙季稻的主產(chǎn)區(qū),而且該區(qū)域雙季早稻的灌漿初期正值7月上、中旬。水稻灌漿初期(揚(yáng)花后第8～14天)是籽粒胚乳細(xì)胞形成和充實(shí)的關(guān)鍵時(shí)期,期間遇高溫?zé)岷?huì)加快胚乳細(xì)胞的分化而增加畸形胚乳細(xì)胞的比例,結(jié)果不利于同化物向胚乳細(xì)胞的運(yùn)輸和積累從而導(dǎo)致水稻籽粒的充實(shí)度下降、粒重降低[7]。同時(shí),高溫影響水稻籽粒細(xì)胞內(nèi)的酶活性,不利于籽粒儲(chǔ)藏物質(zhì)的轉(zhuǎn)化和積累進(jìn)而導(dǎo)致稻米品質(zhì)變劣[8,9]。長(zhǎng)江中下游地區(qū)的雙季早稻是其他作物難以替代的優(yōu)勢(shì)糧食作物,我國(guó)稻谷總產(chǎn)量的幾次大波動(dòng)都與雙季早稻的減產(chǎn)密切相關(guān)。因此,研究水稻灌漿期耐夜間高溫的分子機(jī)制,加快選育對(duì)夜溫升高鈍感的優(yōu)良水稻新品種以保證我國(guó)稻谷產(chǎn)量和品質(zhì)的穩(wěn)定,進(jìn)而對(duì)保障國(guó)家的口糧安全具有重要意義。水稻灌漿期的耐熱性受多基因調(diào)控,其分子調(diào)控機(jī)制相對(duì)復(fù)雜[10,11]。明確哪些基因及其編碼的蛋白質(zhì)參與調(diào)控水稻灌漿期耐熱性是解析其分子機(jī)制的先決內(nèi)容。已有研究表明,灌漿期高溫抑制淀粉支解酶Ⅱb(BEⅡb)活性、增加直鏈淀粉支鏈長(zhǎng)度而減少直鏈淀粉的長(zhǎng)鏈數(shù)量[12];灌漿期高溫提高水稻胚乳細(xì)胞中焦磷酸化酶(AGPase)、可溶性淀粉合成酶(SSS)和淀粉分支酶(SBE)等與淀粉合成有關(guān)的酶活性[13]。在基因表達(dá)方面,灌漿期高溫抑制顆粒淀粉合成酶Ⅰ(GBSSⅠ)、顆粒淀粉合成酶Ⅱb(BEⅡb)、ADP-葡萄糖焦磷酸化酶(包括AGPS2b, AGPS1和AGPL2)、ADP-葡萄糖轉(zhuǎn)運(yùn)體等與淀粉合成相關(guān)基因以及丙酮酸磷酸雙激酶(PPDK)基因的表達(dá)[14,15];灌漿期高溫誘導(dǎo)α-淀粉酶(amylase)基因(包括Amy1A,Amy1C,Amy3 A,Amy3D和Amy3E)表達(dá),加劇胚乳細(xì)胞內(nèi)淀粉的水解而增大籽粒堊白面積[16]。在蛋白質(zhì)表達(dá)方面,灌漿期高溫誘導(dǎo)18．1 k D和17．9 k D熱激蛋白質(zhì)(HSP)、醇溶谷蛋白質(zhì)(Prolamin)和谷蛋白質(zhì)(Glutelin)表達(dá),抑制顆粒淀粉合成酶(granule-boundstarch synthase)、類過(guò)敏原蛋白質(zhì)(allergen-like protein)和延長(zhǎng)因子1β(elongation factor 1β)的表達(dá)[14,17]。在調(diào)控途徑方面,灌漿期夜間高溫誘導(dǎo)線粒體跨膜電子傳遞相關(guān)基因上調(diào)表達(dá)[18],影響三羧酸循環(huán)(TCA)、氨基酸和多胺的生物合成途徑[19]。這些基因和蛋白質(zhì)的鑒定及其表達(dá)模式的明確為進(jìn)一步闡明水稻灌漿期耐熱的分子機(jī)制奠定了研究基礎(chǔ)。然而,水稻在遭遇灌漿期夜間高溫脅迫時(shí),到底哪些基因和蛋白質(zhì)的表達(dá)及其呈現(xiàn)何種表達(dá)模式,其有利于提高水稻對(duì)灌漿期夜間高溫的耐性,尚鮮見報(bào)道[18]。本研究以近等基因系的耐熱水稻純系XN0437T和熱敏感純系XN0437S為供試材料,利用i TRAQ(同位素標(biāo)記相對(duì)和絕對(duì)定量,isobaric tags for relative and absolute quantitation)技術(shù)和高效液相質(zhì)譜檢測(cè)技術(shù)鑒定耐熱和熱敏感水稻純系在應(yīng)答灌漿期夜間高溫過(guò)程中的差異表達(dá)蛋白質(zhì),明確哪些蛋白質(zhì)的上調(diào)表達(dá)或下調(diào)表達(dá)有利于提高水稻灌漿期對(duì)夜間高溫的耐熱性,為進(jìn)一步闡明水稻灌漿期耐夜間高溫的分子機(jī)制奠定研究基礎(chǔ)。

1 材料與方法

1．1 供試水稻材料

供試水稻材料為江西農(nóng)業(yè)大學(xué)早稻灌漿期耐熱抗早衰育種技術(shù)研究課題組選育的一對(duì)近等基因系,即耐熱純系XN0437T和熱敏感純系XN0437S。此對(duì)近等基因系源于協(xié)青早B/N22//協(xié)青早B的回交重組自交系,在江西南昌作為雙季早稻在常規(guī)栽培條件下種植,耐熱純系XN0437T和熱敏感純系XN0437S的株高分別為81．3 cm和81．0 cm,穗長(zhǎng)分別為18．4 cm和18．1 cm,每穗總粒數(shù)分別為94．3粒和95．0粒,全生育期均為106 d。SSR分子標(biāo)記檢測(cè)結(jié)果顯示此對(duì)近等基因系的基因組多態(tài)性僅為1．8%。兩水稻純系于水稻開花后第8天開始進(jìn)行48 h(38.0±0．5)℃高溫處理后,在常溫條件(25.0±0．5)℃下恢復(fù)生長(zhǎng)至成熟并調(diào)查其籽粒充實(shí)度。結(jié)果顯示耐熱水稻純系XN0437T(原名703T)的籽粒充實(shí)度為92．4%,熱敏感純系XN0437S(原名704S)的籽粒充實(shí)度為43．6%,籽粒充實(shí)度差異達(dá)到極顯著水平[20]。

1．2 供試水稻材料的培育、高溫處理及取樣

供試水稻材料的培育、高溫處理及取樣參照Liao等的方法進(jìn)行[18]。水稻的培育采用桶栽的方法,水稻抽穗期、揚(yáng)花期標(biāo)記同一天抽穗且同一天開花的穎花以保證所取樣品的生長(zhǎng)發(fā)育進(jìn)程一致。水稻揚(yáng)花后第8天開始在人工氣候室內(nèi)進(jìn)行高溫處理,夜間溫度設(shè)為38．0℃±0．5℃(高溫處理)和25.0℃±0．5℃(對(duì)照),處理和對(duì)照的白天溫度均設(shè)為(26．0±0．5)℃。暗期時(shí)長(zhǎng)10 h、光期時(shí)長(zhǎng)14 h,連續(xù)處理48 h。設(shè)置3次生物學(xué)重復(fù)。高溫處理結(jié)束時(shí),取處理與對(duì)照的帶標(biāo)記穎花各約5 g,采用錫箔紙包裹,置于液氮中速凍,并于-80℃下保存?zhèn)溆谩?/p>

1．3 水稻籽?？偟鞍踪|(zhì)提取及質(zhì)量檢測(cè)

取水稻籽粒樣品3 g,經(jīng)液氮研磨成粉末后,轉(zhuǎn)移到15 m L離心管中,加入蛋白質(zhì)提取緩沖液(8 mol/L尿素,0．1%SDS,1 mmol/L PMSF,1 mmol/L DTT)于自動(dòng)渦旋混合器上室溫振蕩提取3 h后,4℃、14 000 r/min下離心15 min,取上清液轉(zhuǎn)移至新的15 m L離心管中,加入6倍體積的預(yù)冷丙酮,-20℃下沉淀過(guò)夜。沉淀過(guò)夜樣品于4℃、14 000 r/min下離心15 min,去上清液,將沉淀于4℃真空干燥后獲得總蛋白質(zhì)干粉,-80℃下保存?zhèn)溆谩?/p>

取蛋白質(zhì)干粉50 mg,經(jīng)裂解液(含8 mol/L尿素,2 mol/L硫脲,質(zhì)量分?jǐn)?shù)4%CHAPS,p H3～10體積分?jǐn)?shù)0．5%兩性電解質(zhì),50 mmol/L DTT和1．0 mmol/L PMSF)超聲溶解后,于4℃、14 000 r/ min下離心15 min后,上清液(總蛋白質(zhì)溶液)轉(zhuǎn)移至1．5 m L離心管。采用SDS-PAGE電泳和考馬斯亮藍(lán)染色法檢測(cè)總蛋白質(zhì)的完整性,SDS-PAGE電泳參數(shù)與方法參照Liao等[21]。以牛血清白蛋白質(zhì)為標(biāo)準(zhǔn)品,采用Bradford法測(cè)定蛋白質(zhì)樣品濃度。

1．4 蛋白質(zhì)酶解及iTRAQ標(biāo)記

蛋白質(zhì)樣品先經(jīng)還原和烷基化后,采用胰蛋白質(zhì)酶酶解。每個(gè)樣品取100μg蛋白質(zhì),經(jīng)10 mmol/L二硫蘇糖醇(DTT)于37℃溫浴1 h、55 mmol/L碘乙酰胺于室溫烷化1 h后,加入3．3μg胰蛋白質(zhì)酶于37℃下消化12 h,消化結(jié)束時(shí)加入體積分?jǐn)?shù)1%的蟻酸100μL終止酶解反應(yīng),真空抽干獲得蛋白質(zhì)肽段干粉。蛋白質(zhì)肽段干粉復(fù)溶于8 mol/L尿素(含0．1%SDS)和500 mmol/L三乙基碳酸氫銨的水溶液后,采用AB Sciex公司的8-plex i TRAQ試劑盒進(jìn)行差異顯色標(biāo)記。將試劑盒中8管標(biāo)記試劑(113、114、115、116、117、118、119和121)經(jīng)50μL異丙醇稀釋后分別與相應(yīng)的蛋白質(zhì)肽段干粉復(fù)溶樣品混合,室溫放置1 h,各管中加入100μL去離子水使標(biāo)記物失活,混合8組樣品,凍干備用。

隨機(jī)取3次高溫處理重復(fù)中的2次重復(fù)進(jìn)行i TRAQ分析。耐熱水稻純系的2個(gè)對(duì)照重復(fù)分別命名為TC1和TC2,2個(gè)夜間高溫脅迫重復(fù)分別命名為TT1和TT2,并用iTRAQ試劑113、114、115、116依次進(jìn)行標(biāo)記;熱敏感水稻純系的2個(gè)對(duì)照重復(fù)分別命名為SC1和SC2,2個(gè)夜間高溫脅迫重復(fù)分別命名為ST1和ST2,并用iTRAQ試劑117、118、119、121依次進(jìn)行標(biāo)記。

1．5 iTRAQ標(biāo)記樣品的強(qiáng)陽(yáng)離子交換柱分級(jí)及質(zhì)譜檢測(cè)

iTRAQ標(biāo)記的8個(gè)樣品等量混合后采用C18強(qiáng)陽(yáng)離子柱進(jìn)行樣品分級(jí)。經(jīng)強(qiáng)陽(yáng)離子色譜柱平衡緩沖液A(含p H 2.55、5 mmol/L KH2PO4,體積比為20%的乙腈、H3PO4)平衡25 min后,取標(biāo)記樣品混合物300μL,用強(qiáng)陽(yáng)離子色譜柱平衡緩沖液B (含p H 2.75、5 mmol/L KH2PO4,體積比為20%的乙腈,600 mmol/L KCl,H3PO4)稀釋7倍,正磷酸調(diào)p H值至2.5,離心取上清液進(jìn)行梯度洗脫,每1 min收集1管樣品洗脫液,洗脫液流速為0.2 ml/ min。線性洗脫程序如下:0-3 min,緩沖液B的體積分?jǐn)?shù)由0%升至5%;3-21 min,緩沖液B的體積分?jǐn)?shù)由5%升至20%;21-27 min,緩沖液B的體積分?jǐn)?shù)由20%升至30%;27-33min,緩沖液B的體積分?jǐn)?shù)由30%升至100%。梯度洗脫結(jié)束后,根據(jù)色譜圖的峰形和時(shí)間合并連續(xù)的多肽含量較少的樣品,共形成20個(gè)組分用于肽段的質(zhì)譜檢測(cè)。

質(zhì)譜檢測(cè)采用Thermo Scientific公司生產(chǎn)的Q Exactive質(zhì)譜儀,高效液相系統(tǒng)為戴安NCS3500系統(tǒng)。液相色譜A相為體積分?jǐn)?shù)為99.9%的dd H2O和0.1%的甲酸,B相為體積分?jǐn)?shù)為99.9%的乙腈、0.1%的甲酸。液相色譜洗脫液流速為300 n L/min,液相色譜線性梯度洗脫程序如下:0-5 min,B相體積分?jǐn)?shù)保持3%不變;5-6 min,B相體積分?jǐn)?shù)由3%升至5%;6-36 min,B相體積分?jǐn)?shù)由5%升至20%;36-46 min,B相體積分?jǐn)?shù)由20%升至30%;46-50 min,B相體積分?jǐn)?shù)由30%升至80%,并在之后5 min內(nèi)B相體積分?jǐn)?shù)保持不變;55 -55.5 min,B相體積分?jǐn)?shù)由80%降至3%,并在之后19.5 min內(nèi)B相體積百分?jǐn)?shù)保持3%不變。檢測(cè)質(zhì)譜全掃描范圍m/z＝350～1800,全掃描中選擇離子強(qiáng)度前20位的母離子用標(biāo)準(zhǔn)碰撞能為30 eV的HCD模式碎裂后,進(jìn)行二級(jí)質(zhì)譜序列測(cè)定,以報(bào)告離子的比例進(jìn)行定量。

1．6 蛋白質(zhì)定量及功能注釋

將質(zhì)譜鑒定結(jié)果導(dǎo)入Proteome Discoverer軟件,篩選選擇肽段可信度高(Peptide Confidence＝high)的肽段,并由軟件根據(jù)同一特異肽段(Unique Peptide)在各樣本中的報(bào)告離子峰的面積比值計(jì)算蛋白質(zhì)的表達(dá)量。利用Proteome Discoverer軟件搜索Uniport數(shù)據(jù)庫(kù)(http://ww w．uniprot．org/),注釋蛋白質(zhì)的功能。

1．7 蛋白質(zhì)編碼基因的GO功能和KEGG通路的顯著富集分析

質(zhì)譜數(shù)據(jù)經(jīng)Proteome Discoverer軟件搜索Uniport數(shù)據(jù)庫(kù)鑒定蛋白質(zhì)后,按照蛋白質(zhì)編碼基因ID、利用KOBAS軟件搜索在線GO(Gene ontology)數(shù)據(jù)庫(kù)(http://geneontology．org/),采用Qvalue方法進(jìn)行FDR校正,從而注釋蛋白質(zhì)編碼基因產(chǎn)物屬性和蛋白質(zhì)編碼基因參與的通路并統(tǒng)計(jì)顯著富集的通路。

1．8 差異表達(dá)蛋白質(zhì)分析

根據(jù)軟件Proteome Discoverer計(jì)數(shù)獲得的蛋白質(zhì)表達(dá)量,分2步進(jìn)行差異表達(dá)蛋白質(zhì)分析。首先,依據(jù)蛋白質(zhì)表達(dá)量分析耐熱純系與熱敏感純系各自的夜間高溫脅迫與對(duì)照的差異表達(dá)蛋白質(zhì)(組內(nèi)分析)。若蛋白質(zhì)在夜間高溫脅迫中的表達(dá)量與其在對(duì)照中的表達(dá)量的比值大于1.5倍,則定義為組內(nèi)上調(diào)1.5倍的表達(dá)蛋白質(zhì);上述比值小于0.667(1/1.5),則定義為組內(nèi)下調(diào)1.5倍的表達(dá)蛋白質(zhì)。然后,利用組內(nèi)分析結(jié)果比較耐熱和熱敏感純系之間差異表達(dá)蛋白質(zhì)(組間分析)。以下兩種情況均定義為耐熱純系與熱敏感純系間的差異表達(dá)蛋白質(zhì):1)相同表達(dá)模式的差異蛋白質(zhì):在2個(gè)水稻純系中均呈現(xiàn)相同的上調(diào)或下調(diào)表達(dá)模式,且耐熱純系與熱敏感純系之間的上調(diào)或下調(diào)表達(dá)量的比值大于1.5倍或小于0.667(1/1.5)倍的蛋白質(zhì);2)不同表達(dá)模式的差異蛋白質(zhì),在2個(gè)水稻純系中呈現(xiàn)不同的表達(dá)模式,即在耐熱純系中上調(diào)表達(dá)而在熱敏感純系中下調(diào)表達(dá)的蛋白質(zhì),或在耐熱純系中下調(diào)表達(dá)而在熱敏感純系中上調(diào)表達(dá)的蛋白質(zhì)。

1．9 蛋白質(zhì)編碼基因的mRNA表達(dá)量驗(yàn)證

為了驗(yàn)證iTRAQ數(shù)據(jù)的可靠性,在不同功能分類中選擇了7個(gè)有代表性的差異表達(dá)蛋白質(zhì),根據(jù)其編碼基因在Gen Bank的EST數(shù)據(jù)庫(kù)(w ww．ncbi．nlm．nih．gov/dbEST)中mRNA序列,利用NCBI在線設(shè)計(jì)軟件primer-BLAST設(shè)計(jì)引物,采用熒光定量PCR方法檢測(cè)差異表達(dá)蛋白質(zhì)編碼基因在水稻應(yīng)答灌漿期夜間高溫的表達(dá)量變化。

熒光定量PCR的水稻籽粒樣品來(lái)自“1．2供試水稻材料的培育、高溫處理及取樣”的3次夜間高溫處理重復(fù),樣品的總RNA提取和RNA反轉(zhuǎn)錄合成cDNA第一鏈、qPCR和表達(dá)量計(jì)算參照文獻(xiàn)[18]和[22]的方法進(jìn)行。

2 結(jié)果與分析

2．1 蛋白質(zhì)的鑒定及定量

利用i TRAQ標(biāo)記技術(shù)和液相色譜串聯(lián)質(zhì)譜檢測(cè)技術(shù),供試的8個(gè)樣品共檢測(cè)到肽段次數(shù)為83 254次。通過(guò)軟件Proteome Discoverer過(guò)濾,得到了16 437條高可信度(Peptide Confidence＝high)的肽段,其中可用于蛋白質(zhì)定量的特異肽段為12 911條?？尚烹亩魏吞禺愲亩谓?jīng)Uniport數(shù)據(jù)庫(kù)搜索,最終鑒定并定量了3130個(gè)蛋白質(zhì)。

蛋白質(zhì)的表達(dá)量的差異分析結(jié)果顯示,耐熱水稻純系中上調(diào)1.5倍的蛋白質(zhì)有61個(gè),下調(diào)1.5倍的蛋白質(zhì)有20個(gè)(圖1)。熱敏感水稻純系中上調(diào)1.5倍的蛋白質(zhì)有38個(gè),下調(diào)1.5倍的蛋白質(zhì)有18個(gè)。其中,在耐熱純系和熱敏感純系中均上調(diào)表達(dá)的蛋白質(zhì)有17個(gè),均下調(diào)表達(dá)的蛋白質(zhì)有8個(gè)。

2．2 GO功能和KEGG通路的顯著富集結(jié)果

圖1 耐熱純系與熱敏感純系的應(yīng)答夜間高溫的蛋白質(zhì)數(shù)量Fig．1．Protein amount from the heat-tolerant line and heat-sensitive line responding to high night temperature．

蛋白質(zhì)編碼基因的GO功能(A)和KEGG通路(B)的顯著富集結(jié)果如圖2所示。蛋白質(zhì)編碼基因的GO顯著富集結(jié)果顯示(圖2-A),質(zhì)譜鑒定蛋白質(zhì)的GO功能產(chǎn)物屬性顯著富集(-logP值≥1.5)在生物學(xué)過(guò)程(Biological process)的萜類化合物生物合成過(guò)程(GO ID＝0016114:Terpenoid biosynthetic process)、類異戊二烯生物合成過(guò)程(GO ID＝0008299:Isoprenoid biosynthetic process)、單羧酸生物合成過(guò)程(GO ID＝0072330和0032787: Monocarboxylic acid biosynthetic process)、有機(jī)含羥基化合物生物合成過(guò)程(GO ID＝1901617和1901615:Organic hydroxy compound biosynthetic process)、有機(jī)酸生物合成過(guò)程(GO ID＝0016053: Organic acid biosynthetic process)、羧酸生物合成過(guò)程(GO ID＝0046394:Carboxylic acid biosyntheticprocess)、單一有機(jī)體生物合成過(guò)程(GOID＝0044711:Single-organism biosynthetic process)、脂質(zhì)生物合成過(guò)程(GO ID＝0008610:Lipid biosynthetic process)和小分子生物合成過(guò)程(GO ID＝0044283:Small molecule biosynthetic process);其次,GO功能產(chǎn)物屬性顯著富集(-logP值≥1.5)在細(xì)胞組分(Cellular component)的酶抑制劑活性(GO ID＝0004857:Enzyme inhibitor activity)、肽鏈內(nèi)切酶抑制劑活性(GO ID＝004866:Endopeptidase inhibitor activity)、肽酶調(diào)節(jié)因子活性(GO ID＝0061134:Peptidase regulator activity)、肽酶抑制因子活性(GO ID＝0030414:Peptidase inhibitor activity)、肽鏈內(nèi)切酶調(diào)節(jié)因子活性(GO ID＝0061135:Endopeptidase regulator activity)、絲氨酸蛋白質(zhì)酶抑制劑活性(GO ID＝0004867:Serinetype endopeptidase inhibitor activity)、分子功能調(diào)節(jié)因子(GO ID＝0098772:Molecular function regulator)和酶調(diào)節(jié)因子活性(GO ID＝0030234:Enzyme regulator activity)。蛋白質(zhì)編碼基因的KEGG通路顯著富集結(jié)果顯示(圖2-B),質(zhì)譜鑒定蛋白質(zhì)的KEGG通路顯著富集(-logP值≥4)在內(nèi)質(zhì)網(wǎng)蛋白質(zhì)加工通路上(KEGG ID＝Osa04141: Protein processing in endoplasmic reticulum)。

2．3 差異表達(dá)蛋白質(zhì)的功能注釋及其分類

依據(jù)1.7的差異表達(dá)蛋白質(zhì)判定方法,耐熱水稻純系與熱敏感水稻純系之間存在36個(gè)差異表達(dá)蛋白質(zhì)。這些差異表達(dá)蛋白質(zhì)經(jīng)Uniport數(shù)據(jù)庫(kù)搜索,發(fā)現(xiàn)只有14個(gè)蛋白質(zhì)(占38.9%)有功能注釋、還有12個(gè)蛋白質(zhì)(占33.3%)具有推測(cè)性功能注釋,另有10個(gè)(占27.8%)為功能未知蛋白質(zhì)。14個(gè)有功能注釋的蛋白質(zhì)中(表1),有5個(gè)蛋白質(zhì)(Q9FU69、Q7Y179、Q0IPF8、Q2R1N0和Q84T00)參與能量代謝;有3個(gè)蛋白質(zhì)(Q8GTK2、Q7Y007和Q10BU2)參與物質(zhì)轉(zhuǎn)運(yùn)與代謝;有2個(gè)蛋白質(zhì)(Q2R237和Q658I1)參與光合作用;另外4個(gè)蛋白質(zhì)(Q67TK9、Q10N88、Q84Q72和Q5YLY5)為應(yīng)答逆境的18.1 k D熱激蛋白質(zhì)和鋅指蛋白質(zhì)。

僅有推測(cè)性功能注釋及功能未知的22個(gè)差異表達(dá)蛋白質(zhì)中,在2水稻純系中均呈現(xiàn)上調(diào)表達(dá)1.2倍以上的蛋白質(zhì)有10個(gè)(Q6I587、Q7XI37、Q69MT6、Q338P8、Q7XI22、Q8 H8U1、Q94GQ6、Q0E3F2、Q6YUH8、Q6ES31),均呈現(xiàn)下調(diào)表達(dá)1.2倍以上的蛋白質(zhì)有2個(gè)(Q7XQ85、Q0E225);在耐熱水稻純系中上調(diào)表達(dá)1.2倍以上而在熱敏感水稻純系中下調(diào)表達(dá)1.2倍以上的蛋白質(zhì)有8個(gè)(Q6ZJ47、Q6ZCC9、Q6ZCP8、Q53M16、Q9LWS6、C7JA46、Q0E0B4、Q7XVN6),在耐熱水稻純系中下調(diào)表達(dá)1.2倍以上而在熱敏感水稻純系中上調(diào)表達(dá)1.2倍以上的蛋白質(zhì)有2個(gè)(Q0DTK3、Q5N9C8)。

2．4 差異表達(dá)蛋白質(zhì)的mRNA表達(dá)模式驗(yàn)證

有代表性的7個(gè)差異表達(dá)蛋白質(zhì)及其編碼基因的m RNA表達(dá)模式如圖3所示。通過(guò)iTRAQ差異標(biāo)記結(jié)合蛋白質(zhì)譜分析獲得的蛋白質(zhì)表達(dá)模式與熒光定量PCR中的m RNA表達(dá)模式的趨勢(shì)基本一致,表明通過(guò)i TRAQ差異標(biāo)記結(jié)合高效液相色譜技術(shù)鑒定并定量的蛋白質(zhì)組其結(jié)果可靠。

3 討論

圖2 差異表達(dá)蛋白質(zhì)編碼基因的GO功能(A)和KEGG通路(B)顯著富集結(jié)果Fig．2．Significant enrichment results of Gene Ontology(A)and Kyoto Encyclopedia of Genes(B)．

iTRAQ差異標(biāo)記具有在同一個(gè)體系中同時(shí)標(biāo)記多個(gè)(4或6或8個(gè))樣品的優(yōu)點(diǎn),不僅可以增加后續(xù)質(zhì)譜分析的樣品通量,而且可以減少蛋白質(zhì)色譜分析過(guò)程中的定量誤差[23]。本研究通過(guò)iTRAQ差異標(biāo)記和液相質(zhì)譜技術(shù)鑒定并定量了耐熱與熱敏感水稻近等基因系籽粒中的3130個(gè)蛋白質(zhì)。這些蛋白質(zhì)中,在耐熱與熱敏感水稻純系中相對(duì)表達(dá)量超過(guò)1.5倍的蛋白質(zhì)分別有81個(gè)和56個(gè);進(jìn)一步比較這些蛋白質(zhì)在2個(gè)水稻純系之間的表達(dá)模式差異,結(jié)果發(fā)現(xiàn)耐熱與熱敏感水稻純系之間存在36個(gè)差異表達(dá)蛋白質(zhì)。這些差異表達(dá)蛋白質(zhì)經(jīng)數(shù)據(jù)庫(kù)搜索,發(fā)現(xiàn)僅14個(gè)蛋白質(zhì)已注釋功能,其余蛋白質(zhì)只注釋了推測(cè)性功能或功能未知。這14個(gè)已注釋功能的蛋白質(zhì)中(表1),有5個(gè)蛋白質(zhì)參與能量代謝, 3個(gè)蛋白質(zhì)參與物質(zhì)轉(zhuǎn)運(yùn)與代謝,2個(gè)蛋白質(zhì)參與光合作用,4個(gè)為逆境應(yīng)答蛋白質(zhì)(18.1 k D熱激蛋白質(zhì)和鋅指蛋白質(zhì))。

鋅指蛋白質(zhì)是一類轉(zhuǎn)錄調(diào)控因子蛋白質(zhì),通過(guò)蛋白質(zhì)的鋅、鐵中心與DNA結(jié)合從而調(diào)控基因的表達(dá)[24]。它們不僅在植物生長(zhǎng)發(fā)育中起著重要的基因表達(dá)調(diào)控功能[25,26],而且在植物應(yīng)答生物脅迫和非生物脅迫過(guò)程中扮演著非常重要的調(diào)控基因表達(dá)的角色[27-31]。Chen等[25]的研究報(bào)道鋅指蛋白質(zhì)OsGZF1在水稻種胚發(fā)育過(guò)程中具有調(diào)控籽粒中谷蛋白質(zhì)形成功能。Cao等分析了水稻的Cys2-His2(C2H2)鋅指蛋白質(zhì)家族,發(fā)現(xiàn)其中的20個(gè)鋅指蛋白質(zhì)參與調(diào)控水稻的生長(zhǎng)發(fā)育,28個(gè)參與調(diào)控水稻的授粉受精,22個(gè)參與調(diào)控水稻的抗病性,而且鋅指蛋白質(zhì)VRF1是水稻抗病的不可或缺的調(diào)控因子[32]。Huang等發(fā)現(xiàn)CHY鋅指蛋白質(zhì)具有通過(guò)調(diào)控水稻葉片氣孔的開閉從而提高水稻抗旱性和耐鹽性的功能[27,32]。本研究通過(guò)比較耐熱與熱敏感水稻近等基因系的蛋白質(zhì)組的表達(dá)模式,發(fā)現(xiàn)鋅指蛋白質(zhì)Q67TK9(Zinc knuckle containing proteinlike)和Q10N88(ARF GAP-like zinc finger-containing protein ZIGA2)在耐熱水稻純系中上調(diào)表達(dá)2.3倍以上,而在熱敏感水稻純系中無(wú)明顯表達(dá)變化(＜1.5倍);鋅指蛋白質(zhì)Q5YLY5在耐熱與熱敏感水稻純系中均呈現(xiàn)下調(diào)表達(dá),但是該蛋白質(zhì)在耐熱水稻純系中下調(diào)表達(dá)了4.167倍,而在熱敏感水稻純系中僅下調(diào)表達(dá)1.56倍。表明鋅指蛋白質(zhì)Q67TK9、Q10N88的上調(diào)表達(dá),以及鋅指蛋白質(zhì)Q 5 YLY 5的下調(diào)表達(dá)有利于提高水稻灌漿期對(duì)夜間高溫的耐熱性。

表1 耐熱純系與熱敏感純系的差異表達(dá)蛋白質(zhì)的表達(dá)模式及其功能注釋Table 1．Expression model and functional illustration of the differentially expressed proteins between the heat-tolerant and the heat-sensitive lines．

圖3 差異表達(dá)蛋白質(zhì)及其編碼基因的表達(dá)模式Fig．3．Expression pattern of the differentially expressed protein and its coding gene．

研究表明,籽粒ATP酶促進(jìn)外源營(yíng)養(yǎng)物質(zhì)經(jīng)質(zhì)外體和共質(zhì)體的協(xié)同作用向淀粉胚乳的運(yùn)輸,同時(shí)ATP酶還為胚乳細(xì)胞吸收并積累蛋白質(zhì),進(jìn)一步發(fā)育成蛋白體提供動(dòng)力[33,34]。本研究發(fā)現(xiàn),在耐熱和熱敏感水稻應(yīng)答夜間高溫過(guò)程中,籽粒中與ATP合成過(guò)程相關(guān)的ATP合成酶亞基β(Q0IPF8、LOC_Os12g10570)和ATP酶家族蛋白質(zhì)(Q84T00、LOC_Os03g58800)以及與物質(zhì)運(yùn)轉(zhuǎn)過(guò)程相關(guān)的羧肽酶(Q8GTK2、LOC_Os07g46350)、GDSL脂肪酶或酰基水解酶家族蛋白質(zhì)(Q7Y007、LOC_Os03g47940)和種子萌發(fā)蛋白質(zhì)3-7 (Q10BU2、LOC_Os03g58980)均呈現(xiàn)差異表達(dá)模式。表明夜間高溫影響了ATP酶活性,進(jìn)而阻礙了營(yíng)養(yǎng)物質(zhì)向淀粉胚乳的運(yùn)轉(zhuǎn)和積累。

熱激蛋白質(zhì)普遍存在于動(dòng)植物細(xì)胞內(nèi),它們通過(guò)控制蛋白質(zhì)的折疊和積累從而調(diào)控蛋白質(zhì)表達(dá)的分子或分子伴侶[35]。據(jù)報(bào)道,干旱、高鹽和高溫等環(huán)境條件均可誘導(dǎo)熱激蛋白質(zhì)表達(dá)[36-40]。前期研究中,我們通過(guò)蛋白質(zhì)雙向電泳技術(shù)檢測(cè)了水稻應(yīng)答灌漿期(白天+夜間)高溫的蛋白質(zhì)組[17],發(fā)現(xiàn)18．1 kD熱激蛋白質(zhì)和17．9 kD熱激蛋白質(zhì)在耐熱與熱敏感水稻純系中均呈現(xiàn)上調(diào)表達(dá),而且在熱敏感水稻純系中的上調(diào)表達(dá)倍數(shù)高于其在耐熱水稻純系中的上調(diào)表達(dá)倍數(shù)。本研究利用i TRAQ差異標(biāo)記和液相質(zhì)譜技術(shù)比較耐熱與熱敏感水稻近等基因系應(yīng)答夜間高溫脅迫的蛋白質(zhì)組差異,也檢測(cè)到了18．1 kD熱激蛋白質(zhì)在夜間高溫脅迫與對(duì)照、耐熱與熱敏水稻純系中呈現(xiàn)差異表達(dá),而且該蛋白質(zhì)在熱敏感水稻純系中的上調(diào)表達(dá)倍數(shù)(2．975倍)高于其在耐熱純系中的上調(diào)表達(dá)倍數(shù)(1．906倍)。此外,本研究中還檢測(cè)到17．9 k D熱激蛋白質(zhì)(Q84Q77)在耐熱水稻純系及熱敏感水稻純系中均上調(diào)表達(dá),在耐熱水稻純系中上調(diào)了1．829倍、在熱敏感水稻純系中上調(diào)了2．292倍,但該蛋白質(zhì)在2個(gè)水稻純系間的表達(dá)倍數(shù)差異小于1．5倍。據(jù)此認(rèn)為18．1 kD熱激蛋白質(zhì)的表達(dá)豐度似可以作為水稻對(duì)灌漿期夜間高溫耐熱性的評(píng)判指標(biāo)。

4 結(jié)論

灌漿期夜間高溫影響水稻籽粒細(xì)胞內(nèi)參與能量代謝、物質(zhì)轉(zhuǎn)運(yùn)與代謝、光合作用等相關(guān)蛋白質(zhì)的表達(dá)模式。水稻籽粒細(xì)胞中鋅指蛋白質(zhì)Q67TK9、Q10N88的上調(diào)表達(dá),以及鋅指蛋白質(zhì)Q5YLY5的下調(diào)表達(dá),有利于提高水稻灌漿期對(duì)夜間高溫的耐熱性。

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Analysis on Comparative Proteomics of Rice Grain Between Heat-tolerant and Heat-sensitive Lines Under High Night Temperature Stress at Filling Stage

HUANG Xiaoping1,ZHANG Hongyu2,LEI Gang1,WANG Zhimei1,ZHANG Zhi1,HE Chao1, LIAO Jianglin1,2,?,HUANG Yingjin1,2,?
(1Key Laboratory of Crop Physiology,Ecology and Genetic Breeding,Ministry of Education,Jiangxi Agricultural University, Nanchang 330045;2 Key Laboratory of Agriculture Responding to Climate Change,Jiangxi Agricultural University,Nanchang 330045;?Corresponding author,E-mail:jlliao514815＠163．com;yjhuang_cn＠126．com)

【Objective】To understand molecular mechanism of rice tolerance to high night temperature at filling stage, we identify the differentially expressed proteins(DEPs)and screen the proteins involving in regulating the heat-tolerance in rice．【Method】The heat-tolerant rice line XN0437T and the heat-sensitive rice line XN0437S originated from a near-genetic lines were used as plant materials．Rice was pot-cultivated under traditional management．To ensure that only grain samples with uniform growth were used for proteomes analysis,rice panicle bloomed on the same date were labeled with same markers and spikelets flowering on the same date on the labeled panicle were further labeled．On the 8th day after flowering,rice plants with same label were transferred to chambers for high night temperature treatment．Rice grains with same label were harvested and total proteins of the grain samples were extracted after high temperature treatment．The 8-plex iTRAQ kits combined with the LC-MS/MS technology were used to analyze the DEPs between the heat-tolerant and heat-sensitive lines．【Result】After protein’s databases searching and differentially expressed proteins analysis,3130 proteins were finally identified and 36 proteins showed differential expression levels between the heat-tolerant and heat-sensitive line．For the 36 DEPs,14 proteins(38．9%) had functional annotation,12(33．3%)had putative function,and 10(33．3%)were functional unknown proteins．The 14 function-known proteins were involved in energy metabolism(five proteins),transport and metabolism(three proteins),photosynthesis(two proteins)and defense response(four proteins)．【Conclusion】High night temperature impacts the expressed patterns of the proteins involved in energy metabolism,matter transport and metabolism, photosynthesis and defense response in rice at filling stage．We suggested that up-regulated expression of the zinc finger proteins(Q67TK9 and Q10N88),and down-regulated expression of the zinc finger protein(Q5YLY5)in rice grain could enhance the heat-tolerance in rice．

rice;filling stage;high night temperature;proteomics;iTRAQ technique

Q948.112+.2;S511.01

A

1001-7216(2017)01-0013-10

2016-06-24;修改稿收到日期:2016-09-17。

江西省青年科學(xué)基金重點(diǎn)資助項(xiàng)目(20133 ACB21004);國(guó)家自然科學(xué)基金資助項(xiàng)目(31260315,31471467)。