中国生态农业学报2017，Vol.25Issue(3)：381-390,10.DOI:10.13930/j.cnki.cjea.160581
白菜型冬油菜'陇油7号'叶片响应低温胁迫的差异蛋白鉴定与分析
Differentially expressed proteins in response to low temperature in Brassica campestris 'Long-you No. 7' seedlings
摘要
Abstract
Successful northward expansion of winter rapeseed depends on breeding of super cold-resistant varieties. The 'Longyou' series ofBrassica campestris varieties is cold-resistant, but this mechanism has still not been fully understood. In this study, seedlings of B. campestris 'Long-you No. 7' variety (a strong cold-resistant variety) was used to determine the cold-resistance mechanism at proteomic scale. The TCA (trichloroacetic acid)-acetone precipitation method was used to extract total protein in leaves before and after low temperature stress (4℃ for 7 days). Then the protein extraction method and different pH range of IPG gels was improved and optimized. Furthermore, by using the two-dimensional gel electrophoresis and mass spectrometry methods, the differentially-expressing patterns of total protein in the leaves of 'Long-you No. 7' at five-leaf stage under low temperature stress was determined. The results showed that the average concentration of leaf protein extracted with the improved protein extraction solution containing DDT (DL-Dithiothreitol) and PVPP (crosslinking polyvingypyrrolidone) increased by 3.42μg·μL-1 and the desalting time deceased by 1.14 h. This indicated an improvement in extraction efficiency by the addition of DTT and PVPP to protein extraction solutions. In addition, the addition of protease inhibitor containing PMSF (phenylmethanesulfonyl fluoride) to protein extraction solutions led to the detection of more protein categories; further increasing the number of protein spots by 11.2% (661 versus 587) in the gel pattern. It was also found that by using 17 cm IPG gel (which isolated proteins in a better way) with pH range of 4–7 at vertical electrophoresis stage, higher quality proteomic maps with good repeatability were produced. By using PDQuest 8.0 software, proteomic expression profile of a strong cold-resistant 'Long-you No. 7' seedling before and after low temperature stress was analyzed and a total of 15 differentially-expressed protein spots detected, which was supposedly related to the response to low temperature stress. After further analysis of the protein spots by mass spectrometry, 11 different categories related to low temperature stress were identified, including photosynthesis protein, sugar metabolism protein, material transportation protein and adversity response protein. The findings also showed that the expression level of leaf proteins in 'Long-you No. 7' seedlings differed obviously. Such differentially-expressed proteins were probably critical for cold resistance ofB. campestris, which provided a useful basis for further research.关键词
白菜型冬油菜/陇油7号/低温胁迫/双向电泳/蛋白组学Key words
Brassica campestris/Long-you No. 7/Low temperature stress/Two-dimensional electrophoresis/Proteomics分类
农业科技引用本文复制引用
陈奇,曾秀存,袁金海,刘自刚,孙万仓,方彦,赵新旺,马骊,蒲媛媛,赵艳宁..白菜型冬油菜'陇油7号'叶片响应低温胁迫的差异蛋白鉴定与分析[J].中国生态农业学报,2017,25(3):381-390,10.基金项目
国家重点基础研究计划(973计划)项目(2015CB150206)、国家农业科技成果转化项目(2014G10000317)和甘肃省自然科学基金项目(145RJZG050)资助 The study was supported by the National Key Basic Research Program (2015CB150206), the National Agricultural Science and Technology Achievements Transformation Project (2014G10000317) and the Gansu Provincial Natural Science Foundation Project (145RJZG050). （973计划）
