干旱脅迫下小麥根部蛋白表達變化的雙向電泳分析

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量的主要環境因素之一，研究小麥耐旱機制對提高小麥產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，對其兩葉一心期幼苗在16.7% PEG-6000脅迫0、1、6、72 h的根部蛋白質表達譜進行分析，比較不同脅迫時間點的小麥蛋白質表達譜的差異。結果表明，相對于0 h的表達譜，67種蛋白質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表達2倍以上的20個蛋白質點進行基質輔助激光解吸/電離飛行時間質譜（MALDI-TOF-MS）分析，質譜結果中得到了18個陽性蛋白質點的信息，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、信號轉導和蛋白質合成/代謝等生理生化過程，表明植物在干旱脅迫下調節多種蛋白質的表達，綜合調控其耐旱性。同時，干旱脅迫下未鑒定的豐度差異蛋白質點（DAPs）為克隆新的干旱相關基因和進一步研究小麥的耐旱機理提供了有價值的信息。本試驗結果為進一步研究小麥耐旱機理奠定了基礎。

關鍵詞：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區受到干旱的侵襲[1，2]。克隆耐旱相關基因、研究小麥耐旱機理，對提高小麥在干旱條件下的產量和保證糧食安全有重要意義。

目前，已在小麥中發現多種耐旱相關基因。Xue等[3]從面包小麥中克隆了一個與旱脅迫有關的基因TaNAC69，過表達該基因可以提高轉基因小麥的作物生物量和根長，進而提高轉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答蛋白質（MW：66.2 kD）在128個耐旱等級不同的小麥品系中的表達情況，結果表明該蛋白質的表達量與小麥耐旱性等級呈正相關，說明這個水分脅迫應答蛋白質與小麥的耐旱性密切相關。TaWRKY10的表達量受PEG-6000、NaCl、低溫或過氧化氫處理后上調，過表達可增強轉基因煙草（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，TaODORANT1過表達轉基因煙草在干旱脅迫下有較高的含水量和較低的失水率，這些結果表明TaODORANT1正調控植物的耐旱性[7]。 但是，這些基因功能還不足以全面了解小麥的耐旱機制，挖掘干旱耐受有關的新基因，有利于我們對小麥耐旱機制的研究。

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