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

作　　者：陈奇[1] 袁金海[1] 刘自刚[1] 孙万仓[1] 方彦[1] 赵新旺 马骊[1] 蒲媛媛[1] 赵艳宁 曾秀存[1] 

机构地区：[1]甘肃省油菜工程技术研究中心/甘肃省作物遗传改良与种质创新重点实验室/甘肃省干旱生境作物学重点实验室/甘肃农业大学农学院,兰州730070

出　　处：《中国生态农业学报》2017年第3期381-390,共10页Chinese Journal of Eco-Agriculture

基　　金：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);甘肃省自然科学基金项目(145RJZG050)资助~~

摘　　要：为了从蛋白质组学的角度探讨超强抗寒品种白菜型冬油菜‘陇油7号’的抗寒机理,本研究采用TCA(三氯乙酸)-丙酮沉淀法,提取低温胁迫(4℃,7 d)前后的叶片总蛋白,对蛋白提取方法、IPG(固定pH梯度)胶条种类等环节进行了优化,运用双向电泳和质谱分析技术,鉴定了低温胁迫下‘陇油7号’5叶期叶片总蛋白质组分的表达差异模式。结果表明:改进后的蛋白质提取液(含DTT,二硫苏糖醇)和PVPP(聚乙烯吡咯烷酮)得到的蛋白质平均浓度较改进前高3.42μg·μL-1,除盐时间较改进前短1.14 h;同时,含蛋白酶抑制剂苯甲基磺酰氟(PMSF)的蛋白提取液获得的蛋白质种类丰富,凝胶图谱中可检测到661个蛋白点,较改进前可测蛋白点数(587)提高11.2。采用17 cm p H 4~7的IPG胶条的电泳能更好地分离蛋白,得到重复性好、分辨率高的蛋白质组图谱。利用PDQuest 8.0软件分析比较了超强抗寒品种‘陇油7号’低温胁迫前后的蛋白组表达谱,发现低温胁迫前后共有15个质变的蛋白质点,推测这些差异蛋白点可能与低温胁迫的响应有关。进一步对质变的蛋白质点进行了质谱分析,鉴定出11个与低温胁迫相关的蛋白质点,这些蛋白包括光合作用相关的蛋白、糖代谢相关的蛋白、物质运输相关的蛋白和逆境响应相关的蛋白。而且,低温胁迫处理前后,‘陇油7号’叶片蛋白质的表达水平存在明显差异,这些差异蛋白可能在冬油菜抗寒响应中发挥重要作用。Successful northward expansion of winter rapeseed depends on breeding of super cold-resistant varieties. The ‘Longyou＇ series of Brassica 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 p H 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 3.42 μg·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 p H 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

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 

分 类 号：Q945.78[生物学—植物学] Q946