用焦锑酸钾沉淀法对45℃和28℃下黄瓜(新泰密刺)授粉后柱头及子房中C a2+分布进行了电镜观察,并采用酶联免疫吸附测定法(EL ISA)测定了叶片中的ABA含量及用等电聚焦聚丙烯酰胺双向电泳(IEF-SDS PAGE)方法对其蛋白合成的变化进行了研究....用焦锑酸钾沉淀法对45℃和28℃下黄瓜(新泰密刺)授粉后柱头及子房中C a2+分布进行了电镜观察,并采用酶联免疫吸附测定法(EL ISA)测定了叶片中的ABA含量及用等电聚焦聚丙烯酰胺双向电泳(IEF-SDS PAGE)方法对其蛋白合成的变化进行了研究.结果表明,28℃时柱头中的C a2+主要分布在细胞间隙中,经45℃处理后细胞间隙和胞内C a2+水平均显著升高,胞内外C a2+浓度梯度逐步丧失;45℃处理1 h后柱头和子房中内源ABA含量显著提高;高温处理后柱头和子房中产生了一些新的蛋白质,如柱头中的(MW26.0 kD,P I 5.4)、(MW90.0 kD,P I5.1)、(MW54.0 kD,P I 4.6)、(MW41.0 kD,P I 6.2),子房中的(MW90.0 kD,P I5.2)、(MW61.0 kD,P I 5.4)、(MW48.0 kD,P I 6.4)、(MW40.5 kD,P I 4.9);另有一些蛋白质与常温相比表达量大幅上调,如柱头中的(MW67.0kD,P I 5.8)、(MW56.5 kD,P I 5.8),子房中的(MW70.0 kD,P I 5.7)、(MW57.0 kD,P I 5.7).上述结果表明,C a2+和ABA信号系统均参与了授粉后黄瓜雌性器官对高温胁迫的反应调节并最终导致基因表达发生变化.展开更多
A survey of petal-specific proteomes of soybean(Glycine max(L.) Merr[Non-italic].) was conducted comparing protein expression profiles in different petals. Two-dimensional polyacrylamide gel electrophoresis reference ...A survey of petal-specific proteomes of soybean(Glycine max(L.) Merr[Non-italic].) was conducted comparing protein expression profiles in different petals. Two-dimensional polyacrylamide gel electrophoresis reference maps of protein extracts from standard petals(SP), lateral wings(LW), keel petals(KP), and reproductive organs(RO)(a mixture of stamen and carpel) were obtained. Protein expression in the three petal types was compared using Image Master TM 2 D platinum 6.0 software. This indicated that the proportion of homologous proteins between SP and LW was 59.27%, between SP and KP was 61.48%, and between LW and KP was 60.05%. Within a mass range of 6.5-200.0 ku and pH 4.0-7.0, approximately 590, 646, 544, and 700 protein spots were detected in SP, LW, KP, and RO, respectively. A total of 82 differentially expressed proteins were detected. Sixty-four of these detected spots were differentially expressed and showed more than 2-fold changes in abundance; of these 64 proteins, 26 showed increased expression and 38 showed decreased expression. Among these spots, single organ-specific proteins were also identified.They were ID 49(60.9 ku), ID 45(50.0 ku), and ID 46(40.5 ku) in RO, ID 98(42.0 ku) in SP, and ID 05(29.0 ku) in KP. A total of 14 protein spots from 82 differentially expressed proteins were identified with LC-MS/MS. Further protein identification was conducted using the SwissProt and NCBInr databases. The identified proteins and their putative functions were discussed further. This was the first study reporting the comparison of petal protein profiles of soybean florets using proteomics tools.展开更多
The extraction and solubilization of proteins from seeds of Phaseolus vulgarisms for two-dimension polyacrylamide gel electrophoresis (2D-PAGE) and analysis by mass spectrometry are very sensitive procedures. In this ...The extraction and solubilization of proteins from seeds of Phaseolus vulgarisms for two-dimension polyacrylamide gel electrophoresis (2D-PAGE) and analysis by mass spectrometry are very sensitive procedures. In this study, we used two methods of extraction and solubilization of proteins, the urea/thiourea method and the trichloroacetic acid (TCA)/acetone precipitation method, in order to determine their effectiveness in separating proteins from bean seeds by 2D-PAGE. In both methods, proteins were well separated by 2D PAGE with minor variations in the protein pattern. These two extraction methods showed that it was possible to separate hundreds of very resolvent proteins by 2D electrophoresis. A protein spot was selected on the 2D-PAGE gel, digested with trypsin and analyzed by mass spectrometry (LCMS/ MS). The results suggest that thiourea/urea and TCA methods were effective and reliable for the extraction and solubilization for 2D analysis of proteins from seeds of Phaseolus vulgaris .展开更多
文摘用焦锑酸钾沉淀法对45℃和28℃下黄瓜(新泰密刺)授粉后柱头及子房中C a2+分布进行了电镜观察,并采用酶联免疫吸附测定法(EL ISA)测定了叶片中的ABA含量及用等电聚焦聚丙烯酰胺双向电泳(IEF-SDS PAGE)方法对其蛋白合成的变化进行了研究.结果表明,28℃时柱头中的C a2+主要分布在细胞间隙中,经45℃处理后细胞间隙和胞内C a2+水平均显著升高,胞内外C a2+浓度梯度逐步丧失;45℃处理1 h后柱头和子房中内源ABA含量显著提高;高温处理后柱头和子房中产生了一些新的蛋白质,如柱头中的(MW26.0 kD,P I 5.4)、(MW90.0 kD,P I5.1)、(MW54.0 kD,P I 4.6)、(MW41.0 kD,P I 6.2),子房中的(MW90.0 kD,P I5.2)、(MW61.0 kD,P I 5.4)、(MW48.0 kD,P I 6.4)、(MW40.5 kD,P I 4.9);另有一些蛋白质与常温相比表达量大幅上调,如柱头中的(MW67.0kD,P I 5.8)、(MW56.5 kD,P I 5.8),子房中的(MW70.0 kD,P I 5.7)、(MW57.0 kD,P I 5.7).上述结果表明,C a2+和ABA信号系统均参与了授粉后黄瓜雌性器官对高温胁迫的反应调节并最终导致基因表达发生变化.
基金Supported by Harbin Science and Technology Bureau(2016RQYXJ018,2017RAQXJ104)the Key Laboratory of Soybean Biology in the Chinese Ministry of Education,Northeast Agricultural University(SB17A01)+3 种基金the National Natural Science Foundation of China(31801386)Heilongjiang Natural Science Foundation(LC2018008)Heilongjiang General Young Innovative Talents Training Plan(UNPYSCT-2018158)Certificate of China Postdoctoral Science Foundation Grant(2018M641839)
文摘A survey of petal-specific proteomes of soybean(Glycine max(L.) Merr[Non-italic].) was conducted comparing protein expression profiles in different petals. Two-dimensional polyacrylamide gel electrophoresis reference maps of protein extracts from standard petals(SP), lateral wings(LW), keel petals(KP), and reproductive organs(RO)(a mixture of stamen and carpel) were obtained. Protein expression in the three petal types was compared using Image Master TM 2 D platinum 6.0 software. This indicated that the proportion of homologous proteins between SP and LW was 59.27%, between SP and KP was 61.48%, and between LW and KP was 60.05%. Within a mass range of 6.5-200.0 ku and pH 4.0-7.0, approximately 590, 646, 544, and 700 protein spots were detected in SP, LW, KP, and RO, respectively. A total of 82 differentially expressed proteins were detected. Sixty-four of these detected spots were differentially expressed and showed more than 2-fold changes in abundance; of these 64 proteins, 26 showed increased expression and 38 showed decreased expression. Among these spots, single organ-specific proteins were also identified.They were ID 49(60.9 ku), ID 45(50.0 ku), and ID 46(40.5 ku) in RO, ID 98(42.0 ku) in SP, and ID 05(29.0 ku) in KP. A total of 14 protein spots from 82 differentially expressed proteins were identified with LC-MS/MS. Further protein identification was conducted using the SwissProt and NCBInr databases. The identified proteins and their putative functions were discussed further. This was the first study reporting the comparison of petal protein profiles of soybean florets using proteomics tools.
文摘The extraction and solubilization of proteins from seeds of Phaseolus vulgarisms for two-dimension polyacrylamide gel electrophoresis (2D-PAGE) and analysis by mass spectrometry are very sensitive procedures. In this study, we used two methods of extraction and solubilization of proteins, the urea/thiourea method and the trichloroacetic acid (TCA)/acetone precipitation method, in order to determine their effectiveness in separating proteins from bean seeds by 2D-PAGE. In both methods, proteins were well separated by 2D PAGE with minor variations in the protein pattern. These two extraction methods showed that it was possible to separate hundreds of very resolvent proteins by 2D electrophoresis. A protein spot was selected on the 2D-PAGE gel, digested with trypsin and analyzed by mass spectrometry (LCMS/ MS). The results suggest that thiourea/urea and TCA methods were effective and reliable for the extraction and solubilization for 2D analysis of proteins from seeds of Phaseolus vulgaris .
