鄧艷君 王聰 趙利利 連娟 宋芳媛 劉娜 趙寶存

摘要：干旱是影響小麥生長和產(chǎn)量的主要環(huán)境因素之一，研究小麥耐旱機制對提高小麥產(chǎn)量保證糧食安全有重要的意義。本研究以耐旱小麥晉麥79為材料，利用雙向電泳技術(shù)，對其兩葉一心期幼苗在16.7% PEG-6000脅迫0、1、6、72 h的根部蛋白質(zhì)表達譜進行分析，比較不同脅迫時間點的小麥蛋白質(zhì)表達譜的差異。結(jié)果表明，相對于0 h的表達譜，67種蛋白質(zhì)在不同的脅迫時間點改變了其表達豐度。對其中至少在某一個時間點上調(diào)表達2倍以上的20個蛋白質(zhì)點進行基質(zhì)輔助激光解吸/電離飛行時間質(zhì)譜（MALDI-TOF-MS）分析，質(zhì)譜結(jié)果中得到了18個陽性蛋白質(zhì)點的信息，包括9個功能已知的蛋白和9個未鑒定的蛋白。已知功能的蛋白涉及到能量代謝、脅迫耐受性、信號轉(zhuǎn)導(dǎo)和蛋白質(zhì)合成/代謝等生理生化過程，表明植物在干旱脅迫下調(diào)節(jié)多種蛋白質(zhì)的表達，綜合調(diào)控其耐旱性。同時，干旱脅迫下未鑒定的豐度差異蛋白質(zhì)點（DAPs）為克隆新的干旱相關(guān)基因和進一步研究小麥的耐旱機理提供了有價值的信息。本試驗結(jié)果為進一步研究小麥耐旱機理奠定了基礎(chǔ)。

關(guān)鍵詞：小麥;根部蛋白;表達;耐旱;雙向電泳;MALDI-TOF-MS

中圖分類號：S512.1+1 ?文獻標識號：A ?文章編號：1001-4942（2020）02-0007-08

Abstract Drought stress is one of the major constraints to wheat growth and yield， so studying drought tolerant mechanisms has important significance to wheat yield and food security. In this study， the two-dimensional electrophoresis （2-DE） was used to analyze the protein expression profiles of Jinmai 79 seedling roots exposed to 16.7% PEG-6000 simulated drought stress. The results showed that the expression abundance of 67 proteins changed at different stress time compared with that at 0 h. Moreover， 20 upregulated protein spots， whose abundance levels increased more than 2-fold at a certain time， were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS）. The amino acid sequence information of 18 upregulated protein spots was obtained on the basis of the MS results including 9 reported proteins and 9 not identified proteins. The function reported proteins were involved in several physiological and biochemical pathways such as energy metabolism， stress tolerance， signal transduction， and protein synthesis and metabolism， which showed plant regulated expression of multiple proteins in response to drought stress. The unidentified DAPs under drought stress provided valuable information for cloning novel drought related genes and further studying the drought-tolerant mechanisms of wheat. This study also laid foundations for further research on wheat drought tolerant mechanisms.

Keywords Wheat; Protein in roots; Expression; Drought tolerance; Two-dimensional electrophoresis; MALDI-TOF-MS

小麥是我國主要的糧食作物，隨著水資源危機的加劇，越來越多的小麥生產(chǎn)區(qū)受到干旱的侵襲[1，2]。克隆耐旱相關(guān)基因、研究小麥耐旱機理，對提高小麥在干旱條件下的產(chǎn)量和保證糧食安全有重要意義。

目前，已在小麥中發(fā)現(xiàn)多種耐旱相關(guān)基因。Xue等[3]從面包小麥中克隆了一個與旱脅迫有關(guān)的基因TaNAC69，過表達該基因可以提高轉(zhuǎn)基因小麥的作物生物量和根長，進而提高轉(zhuǎn)基因小麥在干旱脅迫下的存活率。Mao等[4]從小麥中克隆了一個NAC家族的基因TaNAC67，超表達該基因提高了轉(zhuǎn)基因擬南芥對旱、鹽和低溫等非生物脅迫的耐受性，并提高了葉綠素含量、水勢、滲透勢等耐旱相關(guān)生理指標。金秀鋒等[5]利用SDS-PAGE方法檢測了一個水分脅迫應(yīng)答蛋白質(zhì)（MW：66.2 kD）在128個耐旱等級不同的小麥品系中的表達情況，結(jié)果表明該蛋白質(zhì)的表達量與小麥耐旱性等級呈正相關(guān)，說明這個水分脅迫應(yīng)答蛋白質(zhì)與小麥的耐旱性密切相關(guān)。TaWRKY10的表達量受PEG-6000、NaCl、低溫或過氧化氫處理后上調(diào)，過表達可增強轉(zhuǎn)基因煙草（Nicotiana tabacum L.）對干旱和鹽脅迫的耐受性[6]。小麥TaODORANT1在PEG-6000處理時上調(diào)，TaODORANT1過表達轉(zhuǎn)基因煙草在干旱脅迫下有較高的含水量和較低的失水率，這些結(jié)果表明TaODORANT1正調(diào)控植物的耐旱性[7]。 但是，這些基因功能還不足以全面了解小麥的耐旱機制，挖掘干旱耐受有關(guān)的新基因，有利于我們對小麥耐旱機制的研究。

[2] Fahad S， Bajwa A A， Nazir U， et al. Crop production under drought and heat stress： plant responses and management options[J]. Front. Plant Sci.， 2017， 8： 1147.

[3] Xue G P， Way H M， Richardson T， et al. Overexpression of TaNAC69 leads to enhanced transcript levels of stress upregulated genes and dehydration tolerance in bread wheat[J]. Mol. Plant， 2011， 4： 697-712.

[4] Mao X， Chen S， Li A， et al. Novel NAC transcription factor TaNAC67 confers enhanced multi-abiotic stress tolerances in Arabidopsis[J]. PLoS ONE， 2014， 9： 1-15.

[5] 金秀鋒，王憲國，任萬杰，等. 一個水分脅迫應(yīng)答蛋白與小麥抗旱性的關(guān)系及其基因的定位[J]. 作物學(xué)報，2014， 40（2）： 198-204.

[6] Wang C， Deng P， Chen L， et al. Wheat WRKY transcription actor TaWRKY10 confers tolerance to multiple abiotic stresses in transgenic tobacco[J]. PLoS ONE， 2013， 8： e65120.

[7] Wei Q， Luo Q， Wang R， et al. Wheat R2R3-type MYB transcription factor TaODORANT1 positively regulates drought and salt stress responses in transgenic tobacco plants[J]. Front. Plant Sci.， 2017， 8： 1374.

[8] Caruso G， Cavaliere C， Foglia P， et al. Analysis of drought responsive proteins in wheat （Triticum durum） by 2D-PAGE and MALDI-TOF mass spectrometry[J]. Plant Sci.， 2009， 177： 570-576.

[9] Ford K L， Cassin A， Bacic A. Quantitative proteomic analysis of wheat cultivars with differing drought stress tolerance[J]. Front. Plant Sci.， 2011， 2： 44.

[10] Ge P， Ma C， Wang S， et al. Comparative proteomic analysis of grain development in two spring wheat varieties under drought stress[J]. Anal. Bioanal. Chem.， 2012， 402： 1297-1313.

[11] Budak H， Akpinar B A， Unver T， et al.Proteome changes in wild and modern wheat leaves upon drought stress by two-dimensional electrophoresis and nanoLC-ESI-MS/MS[J]. Plant Mol. Biol.， 2013， 83： 89-103.

[12] Peng Z， Wang M， Li F， et al. A proteomic study of the response to salinity and drought stress in an introgression strain of bread wheat[J]. Mol. Cell Proteomics， 2009， 8： 2676-2686.

[13] Damerval C， Vienne D D， Zivy M， et al. Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat seedling proteins[J]. Electrophoresis， 1986， 7： 52-54.

[14] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Anal. Biochem.， 1976， 72： 248-254.

[15] Gao L Y， Wang A L， Li X H， et al. Wheat quality related differential expressions of albumins and globulins revealed by two-dimensional difference gel electrophoresis（2-D DIGE）[J].J. Proteomics， 2009， 73： 279-296.

[16] Valliyodan B， Nguyen H T. Understanding regulatory networks and engineering for enhanced drought tolerance in plants[J]. Plant Biol.， 2006， 9： 189-195.

[17] 樊立強， 劉新月. 國審小麥新品種晉麥79號選育及應(yīng)用研究[J]. 陜西農(nóng)業(yè)科學(xué)， 2009（5）： 8-9，26.

[18] 王鏡巖， 朱圣庚， 徐長法. 生物化學(xué)[M]. 北京： 高等教育出版社， 2006.

[19] 馬莉，陳麗梅. 植物絲氨酸羥甲基轉(zhuǎn)移酶基因研究進展[J]. 生物技術(shù)通報， 2008（2）： 15-19.

[20] 鄧林，陳少良. ATPase與植物抗鹽性[J]. 植物學(xué)通報， 2005， 22（S）： 11-21.

[21] Csiszár J， Gallé A， Horváth E， et al. Different peroxidase activities and expression of abiotic stress-related peroxidases in apical root segments of wheat genotypes with different drought stress tolerance under osmotic stress[J]. Plant Physiol. Biochem.， 2012， 52： 119-129.

[22] Leucci M R， Lenucci M S， Piro G， et al. Water stress and cell wall polysaccharides in the apical root zone of wheat cultivars varying in drought tolerance[J]. J. Plant Physiol.， 2008， 165： 1168-1180.

[23] Mittler R. Oxidative stress， antioxidants and stress tolerance[J]. Trends in Plant Science， 2002， 7： 405-410.

[24] Gill S S， Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J]. Plant Physiol. Biochem.， 2010， 48： 909-930.

[25] Abdrabou A， Brandwein D， Liu C， et al. Rac1 S71 mediates the interaction between Rac1 and 14-3-3 proteins[J]. Cells， 2019， 8： 1006.

[26] Faghani E， Gharechahi J， Komatsu S， et al. Comparative physiology and proteomic analysis of two wheat genotypes contrasting in drought tolerance[J]. J. Proteomics， 2015， 114： 1-15.

[27] Mishra P， Mishra V， Takabe T， et al. Elucidation of salt-tolerance metabolic pathways in contrasting rice genotypes and their segregating progenies[J]. Plant Cell Rep.， 2016， 35：1273-1286.

[28] Trivedi D K， Ansari M W， Tuteja N. Multiple abiotic stress responsive rice cyclophilin （OsCYP-25） mediates a wide range of cellular responses[J]. Commun. Integr. Biol.， 2013， 6： e25260.

[29] Zhao Q， Zhao Y J， Zhao B C， et al. Cloning and functional analysis of wheat V-H+-ATPase subunit genes[J]. Plant Mol. Biol.， 2009， 69： 33-46.