氮素对灌浆期夏玉米叶片蛋白质表达的调控

Regulation of Nitrogen on Protein Expression of Summer Maize(Zea mays L.) Leaves at Filling Stage

【目的】在大田生产条件下研究氮素对灌浆期玉米叶片蛋白质表达的调控。【方法】在大田生产条件下,以紧凑耐密型玉米杂交种登海618为试验材料,研究施氮对玉米穗位叶花后净光合速率、硝酸还原酶(NR)活性、超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性、丙二醛(MDA)含量和可溶性蛋白含量的影响。采用TCA-丙酮沉淀法提取灌浆期(花后20 d)两个施氮处理下玉米穗位叶总蛋白质,并用双向凝胶电泳技术(2-DE)分离获得蛋白质图谱。采用Image Master-2D Elite 7.0图像分析软件对蛋白质图谱进行比较,确定玉米叶片应答灌浆期施氮处理的差异蛋白点。通过MALDI-TOF/TOF MS质谱分析及NCBInr数据库搜索,对差异表达蛋白质进行鉴定并分析其涉及的生物学功能。【结果】开花后,随生育进程的推进,玉米穗位叶净光合速率、叶绿素含量、NR、SOD和POD活性及可溶性蛋白含量均呈下降趋势,而MDA含量则呈上升趋势。相对于不施氮处理,施氮处理下叶片叶绿素含量、NR、SOD和POD活性及可溶性蛋白含量均显著提高,而MDA含量则显著下降。对灌浆期玉米叶片进行双向电泳及图谱分析,分别在施氮和不施氮条件下检测出1 086和1 170个蛋白点。通过图像分析软件进行成对匹配分析,共得到29个显著差异蛋白点。经质谱鉴定分析,29个显著差异蛋白点中有25个被成功鉴定,鉴定成功的蛋白中除未知蛋白(蛋白点55)和30s核糖体蛋白(蛋白点1089)外,其余蛋白表达量均在施氮条件下上调。通过搜索NCBInr数据库,差异表达的蛋白主要分为8类,包括13个能量相关蛋白,2个防御相关蛋白,2个蛋白合成相关蛋白,2个蛋白目的和储存相关蛋白,1个细胞生长相关蛋白,1个次级代谢相关蛋白,1个转运相关蛋白和3个未知蛋白。【结论】施氮对灌浆期玉米叶片光合能力、碳代谢能力、防御能力、蛋白合成能力、蛋白目的和储存能力、以及次级代谢能力等均有显著提升作用。

[Objective] The objective of this experiment is to explore the regulating effect of nitrogen on protein expression of leaf of summer maize under the field production condition. [Method] Denghai 618 was selected as an experimental material, different nitrogen treatments were designed to investigate the photosynthetic rate, the activity of nitrate reductase （NR）, superoxide dismutase （SOD） and peroxidase （POD）, malondialdehyde （MDA） content and soluble protein content in maize ear leaves after tasseling. The total proteins from harvested sample at the stage of 20 days after tasseling were extracted by the trichloroacetate （TCA）-acetone precipitation method and the protein profiles of maize ear leaves were set up by two-dimensional electrophoresis （2-DE）. The proteins in maize leaves responding to nitrogen were identified by ImageMaster-2D Elite 7.0 software. The functions of these differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight massspectrometry （MALDI-TOF/TOF MS） analysis and NCBInr database searching. [Result] After tasseling, the photosynthetic rate, activities of nitrate reductase （NR）, superoxide dismutase （SOD） and peroxidase （POD）, and soluble protein content were significantly decreased, and malondialdehyde （MDA） content was significantly increased. The photosynthetic rate, NR, SOD and POD, and soluble protein content were significantly increased, and MDA content was significantly decreased in nitrogen supply treatment when compared to nitrogen limited treatment. There were 1 086 and 1 170 protein spots were found after 2-DE analysis in nitrogen limited and nitrogen supply treatment, respectively. After a comparative proteomics analysis and MALDI-TOF/TOF mass spectrometry （MS）, 29 differentially expressed proteins were found, including 25 which were successfully identified. They were all up-regulated in nitrogen supply treatment except spots 55 and 1089. They were mainly involved in energy, defense-related pathways, protein synthesis, metabolism, protein transportation and storage, cell growth, secondary metabolism, transcription and unknown. [Conclusion] Nitrogen supply can significantly improve the photosynthesis, carbohydrate catabolism, defense-related pathways, protein synthesis, protein transportation and storage and secondary metabolism in maize leaves at filling stage.