,

(中國(guó)農(nóng)業(yè)科學(xué)院農(nóng)產(chǎn)品加工研究所,北京 100193)

蛋白質(zhì)組學(xué)在葡萄發(fā)育及非生物脅迫中的研究進(jìn)展

方芳,王鳳忠

(中國(guó)農(nóng)業(yè)科學(xué)院農(nóng)產(chǎn)品加工研究所,北京 100193)

葡萄在其生長(zhǎng)發(fā)育過程中極易受到多種非生物因素的影響。蛋白質(zhì)組學(xué)手段可從整體水平上對(duì)生物體內(nèi)的蛋白質(zhì)進(jìn)行系統(tǒng)而全面的研究,在葡萄研究中的應(yīng)用對(duì)于揭示葡萄的發(fā)育及逆境脅迫下的生理和代謝機(jī)制具有重要意義。本文綜述了近年來有關(guān)蛋白質(zhì)組學(xué)手段在葡萄生長(zhǎng)發(fā)育過程和主要非生物脅迫(水分脅迫、溫度脅迫、光照脅迫等)條件下的應(yīng)用和研究進(jìn)展,并對(duì)領(lǐng)域內(nèi)現(xiàn)存問題進(jìn)行分析,對(duì)后續(xù)研究重點(diǎn)進(jìn)行展望,以期為后續(xù)植物發(fā)育生理及逆境生理研究提供參考,為農(nóng)業(yè)生產(chǎn)及農(nóng)作物品質(zhì)提升研究提供理論依據(jù)。

蛋白質(zhì)組學(xué),葡萄,非生物脅迫

蛋白質(zhì)組學(xué)即研究蛋白質(zhì)組的科學(xué),也可定義為研究特定時(shí)間和特定條件下某一生命個(gè)體或組織器官中的全部蛋白質(zhì)的表達(dá)狀況、結(jié)構(gòu)和功能變化的科學(xué)[1-2]。蛋白質(zhì)作為基因功能的具體執(zhí)行者和生命現(xiàn)象的直接體現(xiàn)者[3],決定了蛋白質(zhì)組學(xué)可作為連接基因組學(xué)和代謝組學(xué)的重要橋梁[4]。由于蛋白質(zhì)組學(xué)可以為深入研究生物體內(nèi)復(fù)雜的代謝與調(diào)控機(jī)制提供系統(tǒng)方法和研究平臺(tái)[5],其在近年來得以迅速發(fā)展,并成為繼基因組學(xué)后又一核心和具有廣泛應(yīng)用價(jià)值的科學(xué)[6]。

植物的生長(zhǎng)發(fā)育過程是復(fù)雜的生理生化變化過程,植物在此過程中極易受到多種非生物因素的影響,如光照、極端溫度、干旱、淹水等[7]。植物在感受到外源逆境信號(hào)后,會(huì)迅速進(jìn)行信號(hào)傳導(dǎo)并通過調(diào)節(jié)相關(guān)逆境蛋白表達(dá)來調(diào)整自身的生理狀態(tài),進(jìn)而最大限度降低逆境脅迫的不利影響[6]。蛋白質(zhì)作為信號(hào)轉(zhuǎn)導(dǎo)過程的接收終端,可直觀反映植物體對(duì)外源逆境脅迫的應(yīng)答。因此將蛋白質(zhì)組學(xué)手段應(yīng)用于植物生長(zhǎng)發(fā)育過程及其對(duì)非生物脅迫的響應(yīng)研究,為更加高效、全面而系統(tǒng)地揭示植物的生長(zhǎng)發(fā)育機(jī)制及對(duì)逆境脅迫的應(yīng)答機(jī)制提供了新的可能和途徑。

葡萄是世界上最為重要的水果之一,不僅因其是世界上栽培面積最大、產(chǎn)量最高、消費(fèi)量最大、最具經(jīng)濟(jì)價(jià)值的水果之一[4],還因其是重要的非躍變型果實(shí)代表,常被作為模式植物廣泛用于多種生理及代謝研究[8]。本文以葡萄為研究對(duì)象,對(duì)蛋白質(zhì)組學(xué)在其生長(zhǎng)發(fā)育及非生物脅迫研究中的應(yīng)用進(jìn)行綜述,旨在為植物發(fā)育生理及逆境生理研究提供參考依據(jù)。

1 蛋白質(zhì)組學(xué)在葡萄生長(zhǎng)發(fā)育過程中的應(yīng)用

葡萄果實(shí)的發(fā)育成熟過程是個(gè)復(fù)雜的化學(xué)過程,其與葡萄的糖類、酸類、風(fēng)味物質(zhì)、香氣物質(zhì)及色素等酚類物質(zhì)的形成和積累密切相關(guān),因此將蛋白質(zhì)組學(xué)手段與果實(shí)發(fā)育及成熟過程中的生理生化變化研究有效結(jié)合,對(duì)于全面理解果實(shí)發(fā)育及成熟過程中的變化機(jī)制具有重要意義[9-10]。近年來,蛋白質(zhì)組學(xué)在葡萄植株及葡萄果實(shí)的生長(zhǎng)發(fā)育特性研究中得到了廣泛應(yīng)用[11]。Martínez-Esteso等采用差異凝膠電泳串聯(lián)質(zhì)譜技術(shù)研究玫瑰香葡萄(V.viniferaL. cv. Muscat Hamburg)果實(shí)發(fā)育及成熟過程中的蛋白質(zhì)組變化,在識(shí)別到的156個(gè)蛋白中發(fā)現(xiàn)61個(gè)蛋白在綠果期及成熟期存在差異性表達(dá),并由此推測(cè)綠果期末及成熟早期是葡萄果實(shí)發(fā)育的關(guān)鍵時(shí)期[9]。Kambiranda等通過iTRAQ串聯(lián)質(zhì)譜技術(shù)在研究美國(guó)東南部原葉葡萄(Vitisrotundifoliacv. Noble)果實(shí)發(fā)育及成熟過程中的蛋白質(zhì)組變化時(shí)發(fā)現(xiàn),在檢測(cè)到的674個(gè)蛋白中有76個(gè)蛋白在葡萄果實(shí)發(fā)育過程中呈現(xiàn)差異表達(dá)[12]。次年,Kambiranda等在針對(duì)同一葡萄品種進(jìn)一步研究果實(shí)成熟過程中蛋白質(zhì)組變化時(shí)共檢測(cè)到12組650個(gè)蛋白,其中在果實(shí)成熟過程中出現(xiàn)的差異蛋白主要與碳水化合物的生物合成相關(guān),而在成熟果實(shí)中出現(xiàn)的差異蛋白則多與酚類物質(zhì)和花色苷的生物合成密切相關(guān)[13]。其后,Kambiranda等又以美國(guó)東南部另一原葉葡萄品種(Vitisrotundifoliacv. Carlos)為研究對(duì)象,對(duì)葡萄品種和果實(shí)成熟過程中的蛋白質(zhì)組變化進(jìn)一步研究,發(fā)現(xiàn)葡萄果實(shí)蛋白質(zhì)表達(dá)呈現(xiàn)明顯的發(fā)育階段依賴性,在檢測(cè)到的522個(gè)蛋白中有30個(gè)蛋白與酚類化合物的生物合成密切相關(guān),25個(gè)蛋白與香氣形成密切相關(guān)[10]。Fraige等在研究葡萄品種、成熟度及產(chǎn)區(qū)對(duì)葡萄果實(shí)蛋白質(zhì)組的影響時(shí)發(fā)現(xiàn),在不同產(chǎn)區(qū)不同發(fā)育階段的西拉(Syrah)和赤霞珠(Cabernet Sauvignon)葡萄中可檢測(cè)到差異蛋白128個(gè),其中可識(shí)別蛋白108個(gè),這些差異蛋白主要與糖類物質(zhì)和有機(jī)酸的代謝密切相關(guān),并且可通過蛋白豐度的多變量分析結(jié)果對(duì)葡萄樣品的品種、產(chǎn)區(qū)和發(fā)育階段進(jìn)行了有效區(qū)分[14]。Grimplet等在研究葡萄不同組織部位間的蛋白質(zhì)組表達(dá)差異時(shí),在赤霞珠葡萄果皮和果肉中共檢測(cè)到1047個(gè)蛋白質(zhì),而在果籽中共檢測(cè)到695個(gè)蛋白,可識(shí)別蛋白為163個(gè)。研究結(jié)果表明,赤霞珠葡萄果籽與果皮和果肉中的蛋白質(zhì)組存在明顯差異,果籽中主要以儲(chǔ)存蛋白為主,果皮中主要以苯丙烷類代謝途徑、熱激蛋白及多酚氧化酶為主,果肉中則以基礎(chǔ)能量代謝蛋白為主[11]。由此可見,蛋白質(zhì)組表達(dá)在葡萄生長(zhǎng)發(fā)育過程中呈現(xiàn)明顯的組織間差異性,蛋白質(zhì)組學(xué)技術(shù)在葡萄生長(zhǎng)發(fā)育過程中的應(yīng)用不僅可作為判斷果實(shí)發(fā)育和采收關(guān)鍵時(shí)期的重要依據(jù)[9],同時(shí)可作為有效區(qū)分葡萄品種、產(chǎn)區(qū)和成熟度的重要手段[14]。

2 蛋白質(zhì)組學(xué)在葡萄非生物脅迫中的應(yīng)用

所謂脅迫是指生物體所遭受的不適環(huán)境條件,根據(jù)脅迫類型的不同可分為生物脅迫和非生物脅迫[15],所謂非生物脅迫是指在特定環(huán)境下會(huì)對(duì)生物體造成不適反應(yīng)的全部無生命外源因素的總稱[16]。大量研究表明,過量的非生物脅迫不僅造成葡萄大量減產(chǎn),同時(shí)嚴(yán)重影響葡萄果實(shí)品質(zhì)[17]。但適量的非生物脅迫條件又可有效促進(jìn)次生代謝產(chǎn)物生成,對(duì)促進(jìn)葡萄果實(shí)中某些生物活性物質(zhì)的積累具有重要作用[18-20]。因此近年來有關(guān)非生物脅迫對(duì)葡萄果實(shí)生長(zhǎng)及品質(zhì)調(diào)控領(lǐng)域的研究越來越受關(guān)注,尤其是2007年葡萄全基因組測(cè)序工作完成后,為蛋白質(zhì)組學(xué)手段在葡萄代謝分子機(jī)制及生物學(xué)特性研究方面的應(yīng)用提供了新的可能性[7,11,21-22]。

2.1水分脅迫

水分是影響植物生長(zhǎng)的重要因素,其可通過調(diào)節(jié)葡萄植株的營(yíng)養(yǎng)生長(zhǎng)和生殖生長(zhǎng)間的平衡達(dá)到有效調(diào)節(jié)葡萄果實(shí)產(chǎn)量和質(zhì)量的目的[23-24]。水分的調(diào)節(jié)作用對(duì)于紅葡萄品種尤為重要,葡萄坐果后,通過適度的水分脅迫不僅可有效控制葡萄植株?duì)I養(yǎng)生長(zhǎng)[25],同時(shí)可有效控制葡萄粒徑,進(jìn)而提高果粒受光度,促進(jìn)果實(shí)中的糖類和酸類物質(zhì)的有效積累[26],對(duì)葡萄果實(shí)的品質(zhì)、釀酒特性乃至抗病性等均產(chǎn)生重要影響[27]。前期有關(guān)水分脅迫對(duì)葡萄種植影響的研究多集中在表觀形態(tài)學(xué)及生理生化指標(biāo)變化方面[28],缺乏分子水平上的深入剖析,因此將蛋白質(zhì)組學(xué)技術(shù)手段應(yīng)用于水分脅迫研究,對(duì)于更好地了解水分脅迫條件下葡萄代謝的分子機(jī)制具有重要意義。

近年來蛋白質(zhì)組學(xué)手段在水分脅迫研究中的應(yīng)用也備受關(guān)注。Grimplet等在研究水分缺失條件對(duì)釀酒葡萄果實(shí)的影響時(shí)發(fā)現(xiàn),水分缺失脅迫雖可改變果皮和果肉中約7%的蛋白質(zhì)豐度,但對(duì)果籽中的蛋白質(zhì)表達(dá)幾乎無影響。由此可見,水分缺失脅迫對(duì)赤霞珠葡萄果實(shí)的蛋白質(zhì)組影響存在明顯組織表現(xiàn)特異性[11]。Ghan等在研究坐果至果實(shí)成熟過程中水分缺失脅迫對(duì)葡萄果實(shí)蛋白質(zhì)組的影響時(shí),在3個(gè)紅葡萄品種(赤霞珠、美樂、黑皮諾)和2個(gè)白葡萄品種(霞多麗、賽美蓉)中共識(shí)別出2880個(gè)蛋白,其中有1210個(gè)可定量蛋白,同時(shí)發(fā)現(xiàn)水分缺失脅迫可誘發(fā)不同葡萄品種間的蛋白豐度差異,但在5個(gè)品種間并未發(fā)現(xiàn)共性的脅迫應(yīng)答[29]。Ramesh以美國(guó)東南部2個(gè)雜交葡萄品種為對(duì)象,通過水分脅迫處理來探尋提高葡萄水分利用率的方法,在對(duì)照組和處理組間共檢測(cè)到79個(gè)差異蛋白,并進(jìn)一步證明這些蛋白與葡萄代謝、光合作用、信號(hào)轉(zhuǎn)導(dǎo)、鈣信號(hào)通路及抗氧化體系等密切相關(guān)[27]。上述蛋白質(zhì)組學(xué)研究結(jié)果表明,水分脅迫對(duì)葡萄蛋白質(zhì)組的影響與葡萄的種類、品種密切相關(guān),同時(shí)呈現(xiàn)明顯的發(fā)育階段依賴性和組織表現(xiàn)特異性,適當(dāng)?shù)乃秩笔幚砜勺鳛樘岣吖麑?shí)質(zhì)量及風(fēng)味物質(zhì)含量的重要手段[30-31]。蛋白質(zhì)組學(xué)手段在葡萄水分脅迫研究方面的應(yīng)用可做為優(yōu)化水分脅迫條件,進(jìn)而更好地對(duì)葡萄果實(shí)的產(chǎn)量和品質(zhì)進(jìn)行定向調(diào)控的重要依據(jù)。

2.2溫度脅迫

溫度是影響植物生長(zhǎng)發(fā)育的關(guān)鍵因素,當(dāng)溫度條件明顯超出植物生長(zhǎng)最適范圍時(shí),無論作物的產(chǎn)量還是質(zhì)量都會(huì)受到嚴(yán)重影響[32]。因此,深入了解植物對(duì)溫度耐受性的分子機(jī)制對(duì)于更好地提高植物對(duì)溫度脅迫的適應(yīng)性具有重要意義[33]。葡萄作為溫度敏感性水果其產(chǎn)量和品質(zhì)均與溫度條件密切相關(guān)[34,7]。有關(guān)葡萄植株對(duì)溫度脅迫反應(yīng)的研究前期主要集中在理化特性變化[35]和轉(zhuǎn)錄組學(xué)的研究方面[36],而有關(guān)蛋白質(zhì)組學(xué)的研究較少。Liu等首次以赤霞珠葡萄植株為對(duì)象,以iTRAQ蛋白質(zhì)組學(xué)技術(shù)為手段研究43 ℃高溫下脅迫處理6 h并在25/18 ℃條件下恢復(fù)反應(yīng),葡萄植株的蛋白質(zhì)組學(xué)變化。在高溫脅迫及恢復(fù)反應(yīng)過程中,共發(fā)現(xiàn)174個(gè)差異蛋白,其中脅迫條件和恢復(fù)處理過程中同時(shí)出現(xiàn)的蛋白有42個(gè),熱脅迫過程中出現(xiàn)的特異蛋白為113個(gè),恢復(fù)處理過程中出現(xiàn)的特異蛋白為103個(gè),檢出的蛋白中有20%與光合作用相關(guān),另有8%的蛋白質(zhì)與逆境脅迫密切相關(guān)[33]。George等在研究高溫脅迫(34、42 ℃)和低溫脅迫(18、10 ℃)對(duì)赤霞珠葡萄果實(shí)細(xì)胞蛋白質(zhì)組的影響時(shí)發(fā)現(xiàn),在檢測(cè)到的2042個(gè)蛋白中有55個(gè)蛋白僅在42 ℃極端高溫脅迫條件下有檢出,53個(gè)蛋白僅在10 ℃極端低溫脅迫條件下有檢出,苯丙烷類代謝途徑中的9個(gè)蛋白僅在極端低溫脅迫條件下表現(xiàn)為豐度升高,進(jìn)而推斷其可能是冷應(yīng)激蛋白[22]。此外大量研究還表明,熱處理在保持果實(shí)品質(zhì)、延長(zhǎng)貨架期及減少果實(shí)冷害損傷方面具有重要作用[37-38]。Wu等通過蛋白質(zhì)組學(xué)手段研究葡萄儲(chǔ)藏過程中熱激處理對(duì)果實(shí)品質(zhì)的影響及其分子機(jī)制。研究結(jié)果顯示熱處理組與對(duì)照組間存在64個(gè)差異蛋白,其中大多數(shù)上調(diào)蛋白與葡萄果實(shí)的防御反應(yīng)和氧化還原代謝、碳水化合物代謝及能量代謝相關(guān)[39]。由此可見,將蛋白質(zhì)組學(xué)手段應(yīng)用于葡萄果實(shí)對(duì)溫度脅迫反應(yīng)應(yīng)答方面的研究，對(duì)于更好更全面地了解其逆境脅迫分子機(jī)制具有重要意義,但目前在此領(lǐng)域的研究與應(yīng)用較少[22],盡管有關(guān)溫度脅迫對(duì)葡萄植株的影響已在轉(zhuǎn)錄組學(xué)方面有報(bào)道[40],但轉(zhuǎn)錄組學(xué)研究結(jié)果未必與蛋白質(zhì)組學(xué)研究結(jié)果完全吻合[41],后續(xù)的研究工作中有關(guān)蛋白質(zhì)組層面的研究還需進(jìn)一步加強(qiáng)。

2.3光照脅迫

光照是植物生長(zhǎng)的必備條件,其與植物的生長(zhǎng)、發(fā)育、開花、結(jié)果及果實(shí)發(fā)育等多種生理反應(yīng)密切相關(guān)[42-43]。適量的光照不僅可有效促進(jìn)植物的營(yíng)養(yǎng)生長(zhǎng)及生殖生長(zhǎng),同時(shí)對(duì)植物次生代謝產(chǎn)物積累具有重要的調(diào)節(jié)作用。但不適當(dāng)?shù)墓庹仗幚?則可造成植物體損傷,因此研究葡萄對(duì)光照脅迫的反應(yīng),尤其采用蛋白質(zhì)組學(xué)手段對(duì)其分子機(jī)制進(jìn)行探索,對(duì)于定向調(diào)控葡萄果實(shí)次生代謝產(chǎn)物生成,進(jìn)而提高葡萄果實(shí)及葡萄酒的生物活性物質(zhì)含量具有重要作用。尤其近年來臭氧層破壞嚴(yán)重導(dǎo)致地表紫外輻射增強(qiáng),勢(shì)必對(duì)地表生物造成損傷,有關(guān)植物對(duì)光照脅迫的應(yīng)答反應(yīng)越發(fā)引起科學(xué)界廣泛關(guān)注。大量研究表明植物對(duì)UV等光照脅迫的反應(yīng)與植物品種、植株年齡、緯度、輻照水平、輻照波長(zhǎng)及輻照時(shí)間等密切相關(guān)[44-46]。目前蛋白質(zhì)組學(xué)手段已經(jīng)在玉米[47]、水稻[48]及金銀花[49]等多種植物的光照脅迫研究中得到應(yīng)用,但在葡萄中的應(yīng)用較少。前期有關(guān)光照脅迫條件下葡萄應(yīng)答反應(yīng)的研究主要集中在植物表觀變化研究[46]、生理生化研究及次生代謝產(chǎn)物定向積累研究[50-52]等方面,代謝組學(xué)和轉(zhuǎn)錄組學(xué)手段在此過程中也得到了應(yīng)用[53],但目前有關(guān)蛋白質(zhì)組層面的研究極少。Niu等以Jingxiu葡萄(Vitis vinifera)為對(duì)象,研究日光缺失對(duì)葡萄果實(shí)蛋白質(zhì)組的影響,發(fā)現(xiàn)在檢測(cè)到的1500多個(gè)蛋白質(zhì)中,正常光照組和日光缺失組中有96個(gè)蛋白質(zhì)的積累模式存在明顯差異。鑒定出的72個(gè)蛋白質(zhì)中包括35個(gè)下調(diào)蛋白和37個(gè)上調(diào)蛋白,其中在日光缺失條件下與光合作用和次生代謝產(chǎn)物合成相關(guān)的蛋白質(zhì)積累明顯減少,但與能量供給、糖酵解和三羧酸循環(huán)相關(guān)的蛋白卻出現(xiàn)大量積累[21]。上述研究不僅在蛋白質(zhì)組層面為日光缺失對(duì)葡萄果實(shí)的影響提供了數(shù)據(jù)支撐,同時(shí)也為蛋白質(zhì)組學(xué)手段在葡萄代謝生理研究中的應(yīng)用提供了新的可能。

3 結(jié)論與展望

葡萄的生長(zhǎng)發(fā)育過程及對(duì)非生物脅迫的逆境應(yīng)答反應(yīng)是個(gè)非常復(fù)雜的生理生化過程,在此過程中,葡萄初生及次生代謝途徑中的相關(guān)酶被特異激活或抑制,進(jìn)而幫助果實(shí)實(shí)現(xiàn)正常的生長(zhǎng)發(fā)育或協(xié)助增強(qiáng)其對(duì)外源脅迫的防御能力,因此,了解植物生長(zhǎng)發(fā)育及在非生物脅迫條件下的逆境應(yīng)答分子機(jī)制對(duì)于提高葡萄抗逆性,更好地指導(dǎo)葡萄生產(chǎn)具有重要意義。蛋白質(zhì)組學(xué)作為一種新興的高通量蛋白質(zhì)研究手段,雖然在多物種上已得到應(yīng)用,但目前在葡萄非生物脅迫應(yīng)答機(jī)制領(lǐng)域的應(yīng)用極其有限,蛋白質(zhì)層面研究的局限不僅在一定程度上限制了葡萄品質(zhì)提升,同時(shí)也制約了葡萄的栽培和生產(chǎn)。

針對(duì)上述問題,未來有關(guān)葡萄生長(zhǎng)發(fā)育及逆境脅迫應(yīng)答反應(yīng)的研究重心應(yīng)逐漸從葡萄表觀遺傳學(xué)及代謝物消長(zhǎng)規(guī)律研究向逆境應(yīng)答分子機(jī)制方向轉(zhuǎn)變,在加強(qiáng)蛋白質(zhì)組學(xué)手段應(yīng)用的同時(shí),注意加強(qiáng)與基因組學(xué)、代謝組學(xué)等多組學(xué)手段結(jié)合將成為未來發(fā)展的必然趨勢(shì)。

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Researchprogressofproteomicsingrapesduringgrapeberrydevelopmentandunderabioticstress

FANGFang,WANGFeng-zhong

(Institute of Food Science and Technology,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

Grape is vulnerable to various external abiotic factors during grape berry development. Proteomics is a powerful tool to study proteins from large scale. The application of proteomics in grape research will help to clarify the physiological and metabolic mechanism under abiotic stresses. In this paper,the latest researches progress of proteomics in grapes during grape berry development and under major abiotic stresses(water deficit,temperature stress and light stress)were reviewed. The potential problems in the research field were discussed,and the research emphasis in the future was suggested,in order to provide reference for the future studies on plant development physiology and stress physiology,and provide theoretical basis for agricultural production and crop quality improvement.

proteomics;grape;abiotic stress

2017-02-07

方芳(1980-)，女，博士，副研究員，研究方向：植物源食品功能活性物質(zhì)挖掘與代謝調(diào)控研究，E-mail:fangfang9992@126.com。

*通訊作者:王鳳忠(1972-)，男，博士，研究員，研究方向：農(nóng)產(chǎn)品功能因子研究與利用，E-mail:wangfengzhong@sina.com。

國(guó)家自然科學(xué)基金項(xiàng)目(31401823)。

TS255

:A

:1002-0306(2017)16-0330-05

10.13386/j.issn1002-0306.2017.16.062