小麦叶片胞间隙水分胁迫诱导蛋白的分离、纯化及质谱鉴定
摘要
细胞间隙液(IWF)中存在许多与植物生长发育相关的物质,为研究植物在逆境胁迫下所发生的生理生化反应提供了重要的资料。本文以“小偃6号”为材料,采用真空渗入离心法,通过设定不同的离心力和时间,提取小麦叶片胞间隙液。提取的胞间隙液经过SDS-PAGE电泳后,用兔抗Rubisco抗体进行Western-blotting试验。结果表明,在离心力为3000g,时间为10,15和20min所提的胞间隙液中均检测到Rubisco大亚基的特异条带。说明在此条件下提取的胞间隙液有细胞器或其它胞内物质的污染。因此,结合其产出量,小麦叶片胞间隙液提取的离心条件应选择为1000-2500g,15-20min。
2周龄小麦幼苗用不同浓度PEG-6000模拟干旱处理,1000g,20min提取小麦叶片胞间隙液。测定胁迫2,4,6,8d和复水2d的小麦叶片及胞间隙液中POD、PPO、PAL及几丁质酶的活性,结果表明:随着胁迫强度的增加和处理时间的延长,POD、PPO、PAL(胞间隙除外)、几丁质酶活性均表现上升的趋势,复水2d后下降,但各项指标均没有恢复到对照水平。通过比较胞内、外酶活性大小发现,POD,PAL和PPO活性(处理第2-4d)胞内酶活大于胞间隙,几丁质酶和PPO(第6-8d)则相反。
对27.5%PEG处理后的小麦叶片胞间隙液进行SDS-PAGE电泳分析,结果表明,和对照相比,27.5%PEG处理第4,6,8d小麦叶片胞间隙出现分子量约为50kD,36kD和21kD的三种差异蛋白,在处理第4d开始表达,且复水2d后消失,推测这三种蛋白是水分诱导产生的与小麦干旱抗性相关。
用电洗脱的方法对50kD蛋白进行纯化,通过MALDI-TOF-MS(基质辅助激光解析电离飞行时间质谱)分析,将所得的PMF(肽指纹图谱)在NCBInr蛋白质数据库中比对,发现的50kD蛋白是一种未知蛋白,有待进一步研究和分析。
The intercellular washing fluids (IWF) contain many components concerned with the growth and development of plants, which provide some valuable materials for further study of plant physiological and biochemical responses to adverse circumstances. In this study, primary leaves of wheat were detached, vacuum infiltrated with distilled water and centrifuged on various centrifugal force and time to extract IWF, whose purity was then analyzed by SDS-PAGE and Western-blotting with rabbit-anti-Rubisco antibody. The result indicated that the large subunit of Rubisco was detected when the centrifugal force was 3000g, lasting for 10-20minutes. This showed that the extracted IWF were not pure on this or higher than this centrifugation condition. Therefore, combined with its extraction volumes, the centrifugal condition of IWF from wheat leaves should be between 1000-2500g, 15-20min.
In this experiment, Wheat xiaoyan6 was used as sample, whose seedlings were treated with different concentrations of PEG-6000 solution, then the IWF of wheat leaves were extracted at 1000g, 20min. Activities of POD, PPO, PAL and chitinase were determined 2, 4, 6 and 8 days after treatment and 2 days after rewatering. The results showed that as the increase of stress intensity and the prolongation of treatment time, except the activities PAL of IWF, the activities of POD, PPO, PAL and chitinase of leaves and IWF showed a tendency of being increased at first, then decreased after rehydration, however, none index recovered to the level of CK. After comparation of the activities of these indexes in cells and IWF, we found that the activities of POD, PAL and PPO(2-4 days after treatment) were higher in cells than in IWF, while the activities of chitinase and PPO(4-8 days after treatment ) led to the opposite results.
The proteins of IWF were extracted and then separated by SDS-PAGE, the results showed that three subunits, whose molecular weight were about 50kD, 36kD and 21kD, were induced 4, 6 and 8 days after treatment with 27.5%PEG, then disappeared 2 days after rewatering. Thus, in conclution, three water stress induced proteins had a correlation with the response of wheat to drought stress.
The 50kD protein was purified by electro elution protocol and was analyzed by MALDI-TOF-MS. Its peptide mass fingerprinting(PMF)result was searched in protein data bank, which demonstrated no similar proteins reported previously. Therefore, the further identification should be done in the following.
2周龄小麦幼苗用不同浓度PEG-6000模拟干旱处理,1000g,20min提取小麦叶片胞间隙液。测定胁迫2,4,6,8d和复水2d的小麦叶片及胞间隙液中POD、PPO、PAL及几丁质酶的活性,结果表明:随着胁迫强度的增加和处理时间的延长,POD、PPO、PAL(胞间隙除外)、几丁质酶活性均表现上升的趋势,复水2d后下降,但各项指标均没有恢复到对照水平。通过比较胞内、外酶活性大小发现,POD,PAL和PPO活性(处理第2-4d)胞内酶活大于胞间隙,几丁质酶和PPO(第6-8d)则相反。
对27.5%PEG处理后的小麦叶片胞间隙液进行SDS-PAGE电泳分析,结果表明,和对照相比,27.5%PEG处理第4,6,8d小麦叶片胞间隙出现分子量约为50kD,36kD和21kD的三种差异蛋白,在处理第4d开始表达,且复水2d后消失,推测这三种蛋白是水分诱导产生的与小麦干旱抗性相关。
用电洗脱的方法对50kD蛋白进行纯化,通过MALDI-TOF-MS(基质辅助激光解析电离飞行时间质谱)分析,将所得的PMF(肽指纹图谱)在NCBInr蛋白质数据库中比对,发现的50kD蛋白是一种未知蛋白,有待进一步研究和分析。
The intercellular washing fluids (IWF) contain many components concerned with the growth and development of plants, which provide some valuable materials for further study of plant physiological and biochemical responses to adverse circumstances. In this study, primary leaves of wheat were detached, vacuum infiltrated with distilled water and centrifuged on various centrifugal force and time to extract IWF, whose purity was then analyzed by SDS-PAGE and Western-blotting with rabbit-anti-Rubisco antibody. The result indicated that the large subunit of Rubisco was detected when the centrifugal force was 3000g, lasting for 10-20minutes. This showed that the extracted IWF were not pure on this or higher than this centrifugation condition. Therefore, combined with its extraction volumes, the centrifugal condition of IWF from wheat leaves should be between 1000-2500g, 15-20min.
In this experiment, Wheat xiaoyan6 was used as sample, whose seedlings were treated with different concentrations of PEG-6000 solution, then the IWF of wheat leaves were extracted at 1000g, 20min. Activities of POD, PPO, PAL and chitinase were determined 2, 4, 6 and 8 days after treatment and 2 days after rewatering. The results showed that as the increase of stress intensity and the prolongation of treatment time, except the activities PAL of IWF, the activities of POD, PPO, PAL and chitinase of leaves and IWF showed a tendency of being increased at first, then decreased after rehydration, however, none index recovered to the level of CK. After comparation of the activities of these indexes in cells and IWF, we found that the activities of POD, PAL and PPO(2-4 days after treatment) were higher in cells than in IWF, while the activities of chitinase and PPO(4-8 days after treatment ) led to the opposite results.
The proteins of IWF were extracted and then separated by SDS-PAGE, the results showed that three subunits, whose molecular weight were about 50kD, 36kD and 21kD, were induced 4, 6 and 8 days after treatment with 27.5%PEG, then disappeared 2 days after rewatering. Thus, in conclution, three water stress induced proteins had a correlation with the response of wheat to drought stress.
The 50kD protein was purified by electro elution protocol and was analyzed by MALDI-TOF-MS. Its peptide mass fingerprinting(PMF)result was searched in protein data bank, which demonstrated no similar proteins reported previously. Therefore, the further identification should be done in the following.
引文
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