基于iTRAQ技术的干旱胁迫下玉米苗期蛋白质组学研究
【目的】从蛋白质组学的层面研究玉米苗期干旱胁迫后蛋白表达丰度的变化,了解玉米苗期耐旱响应过程中所涉及的代谢途径,探讨相关分子调控机理。【方法】以B73玉米幼苗为供试材料,在人工气候箱中培养至三叶一心期,对照组正常供水,处理组则停止供水,5天后同时取材,并利用酚抽提法提取总蛋白。采用同位素标记相对定量(iTRAQ)技术进行蛋白质组学分析。通过匹配Uniprot玉米蛋白数据库鉴定蛋白,并分析鉴定干旱胁迫后丰度发生了显著变化的蛋白,进而将其分类以探讨耐旱响应机制。【结果】通过匹配玉米蛋白数据库及定量分析,共检测到玉米幼苗中的207个蛋白在干旱胁迫后发生了显著的丰度变化。根据蛋白注释情况,进一步对其中142个蛋白进行了更为细致的归类分析,根据蛋白的功能我们将这些蛋白分为10类,包括：信号传导(7个)、渗透调节(5个)、蛋白合成与折叠(31个)、ROS清除(14个)、膜运输(19个)、转录相关(19个)、细胞结构与细胞周期(9个)、脂肪酸代谢(3个)、碳水化合物与能量代谢(15个)、光合作用与光呼吸(20个)。综合分析发现玉米苗期对干旱胁迫的响应涉及了诸多过程,涉及光反应和呼气作用的差异蛋白多表现为丰度上升；涉及碳水化合物及蛋白质合成差异蛋白多表现为丰度下降；与渗透调节相关的脱水蛋白、脯氨酸代谢和渗透胁迫相关的蛋白酶则显示为丰度上升。另外,干旱胁迫还能导致植物体内活性氧大量产生,活性氧清除相关的酶类也会发生明显的丰度上升。【结论】干旱胁迫下B73幼苗中丰度发生显著变化的蛋白涉及多个代谢途径,构建了一个协同调控响应机制,我们推测玉米苗期主要通过降低植株生长速率、减少水分散失、清除自由基等多种方式维持其在干旱胁迫条件下的生长发育过程。
英文摘要:
【Objective】To investigate the molecular mechanisms of drought tolerance response in maize seedlings, protein abundance changes were studied by using approach of proteomics. 【Method】 Maize inbred line B73 was used as experimental material. The seedling stage drought treatment was conducted in an artificial climatic chamber and the plant material was obtained after 5 days of drought treatment. Total protein was extracted by phenol extraction method and proteomics analysis was performed using isotopic tagging relative quantitation (iTRAQ). The proteins were identified by matching Uniprot maize protein database, and the proteins that were significantly changed under the drought stress were analyzed and classified to understanding the drought response mechanism. 【Results】By matching the database and quantitative analysis, a total of 207 proteins were detected to be significantly differently expressed during drought tolerance in maize seedlings. According to the protein annotation, 142 proteins were further categorized and analyzed in details. These differential proteins were divided into 10 categories including: signaling (7 proteins), osmoregulation (5 proteins), protein synthesis and folding (31 proteins), ROS clearance (14 proteins), membrane transport (19 proteins), transcription related (19 proteins), cell structure and cell cycle (9 proteins), fatty acid metabolism (3 proteins), carbohydrate and energy metabolism (15 proteins), as well as photosynthesis and photorespiration (20 proteins). Many pathways and proteins were involved in response to drought stress at maize seedling stage. most proteins involved in photoreaction and exhalation were accumulated; most of carbohydrate and protein synthesis related proteins were reduced; and proteins associated with osmotic adjustment such as dehydrins, proline, and osmotic stress were accumulated. In addition, drought stress always induces excessive reactive oxygen species (ROS) in plants, and the significant accumulation of some ROS scavenging related enzymes was detected in this study. 【Conclusion】Under drought stress, the abundance of many proteins was significantly changed, and these proteins were involved in a variety of metabolic pathways. Comprehensive analysis of the results, we speculated that maize seedlings might maintain the growth and development process under drought stress conditions by decreasing plant growth rate, reducing water loss and scavenging free radicals.
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