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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。
In order to explore the cold resistance mechanism of super cold-resistant winter rapeseed (Longyou No.7) from proteomics perspective, TCA (trichloroacetic acid) -acetone precipitation method was used to extract low temperature stress (4 ℃, 7 d) The total protein in leaves before and after was optimized for protein extraction method, IPG (fixed pH gradient) strip type and so on. The two-dimensional gel electrophoresis and mass spectrometry were used to identify the 5-leaf stage of ’Longyou No.7’ Expression patterns of total protein components in leaves. The results showed that the average protein concentration of the improved protein extract (containing DTT, dithiothreitol) and PVPP (polyvinylpyrrolidone) was 3.42 μg · μL-1 higher than that before the improvement and the desalting time was 1.14 h. At the same time, PMSF protein extract obtained a rich variety of proteins. In the gel chromatogram, 661 protein spots were detected, which was significantly higher than that of the pre-measurable protein spots (587) 11.2. Electrophoresis of IPG strips with 17 cm p H 4 ~ 7 provided better resolution of the proteome and resulted in a more reproducible and highly resolved proteome map. PDQuest 8.0 software was used to analyze and compare the proteome expression profiles of super cold-resistant variety Longyou 7 before and after low temperature stress. It was found that there were 15 qualitative and quantitative protein spots before and after low temperature stress. It is speculated that these differences may be related to low temperature stress response related. Further mass spectrometry analysis of the qualitatively altered protein spots identified eleven spots associated with low temperature stress. These proteins include photosynthesis related proteins, glycometabolism related proteins, transport proteins and stress response related proteins. Moreover, there was a significant difference in the protein expression levels in ’Longyou 7’ before and after low temperature stress treatment, and these differential proteins may play an important role in the cold tolerance of winter rape.