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为了研究质外体蛋白在低温胁迫下白菜型冬油菜抗寒的作用机理,以陇油7号五叶期叶片为试材,采用2种不同提取液(提取液A:0.1 mol/L p H值=7.6 Tris-HCl、0.2 mol/L KCl、1 mmol/L PMSF,提取液B:0.05 mol/L p H值=7.6 Tris-HCl、0.01 mol/L EDTA-Na2、0.02 mol/L Vc、1 mmol/L PMSF)进行质外体蛋白提取效果比较。结果表明:提取液A的蛋白提取率达到了(0.073±0.004 6)mg/g,比提取液B提高了42.88%;经六磷酸葡萄糖脱氢酶(G6PDH)活性检测,提取液A提取的蛋白质污染率较提取液B低0.91%;双向电泳第一向等电聚焦IEF中,提取液A提取的蛋白质不仅除盐时间较提取液B短4.5 h,且获得的凝胶图谱清晰;通过丰度计算,得到陇油7号低温胁迫(4℃,48 h)后具有显著差异的蛋白点339个,其中表达量变化在2倍以上的蛋白点为46个,包括上调表达蛋白点18个,下调表达蛋白点21个,特异性表达蛋白点7个,推测这些差异蛋白点可能与低温胁迫有关。对特异表达的7个蛋白点进行质谱分析鉴定,得到与低温胁迫相关的蛋白,为白菜型冬油菜超强抗寒品种陇油7号抗寒相关分子机理的研究奠定了基础理论。
In order to study the mechanism of cold resistance of prokaryotic protein in cabbage type Brassica napus under low temperature stress, taking two leaves of Longyou No.5 leaf stage as test material, two different extracts (extract A: 0.1 mol / L p H Value = 7.6 Tris-HCl, 0.2 mol / L KCl, and 1 mmol / L PMSF. Extract B: 0.05 mol / L p H = 7.6 Tris-HCl, 0.01 mol / L EDTA-Na2, 0.02 mol / L Vc, 1 mmol / L PMSF). The results showed that the protein extraction rate of extract A reached 0.073 ± 0.0046 mg / g, which was 42.88% higher than that of extract B. The protein extracted from liquid A was detected by G6PDH activity assay The contamination rate was 0.91% lower than that of the extract B. In the first isoelectric focusing IEF, the protein extracted by the extract A not only had the desalting time 4.5 hours shorter than that of the extract B, but also the gel map obtained was clear. The results showed that there were 339 protein spots with significant difference after long-time cold stress (4 ℃, 48 h) in Longyou No.7, among them, there were 46 protein spots whose expression changed more than 2 folds, including 18 spots with up-regulated expression There were 21 expressed protein spots and 7 specific expressed protein spots, suggesting that these differential protein spots may be related to low temperature stress. Seven specific protein spots were identified by mass spectrometry, and the proteins related to low temperature stress were obtained, which laid the foundation for the research on molecular mechanism of cold resistance in Brassica napus L.