适用于蛋白质组分析的粉红单端孢菌蛋白提取方法的建立
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摘要
为了优化一种适用于粉红单端孢菌双向电泳的蛋白质提取方法,以期为蛋白质组学水平研究粉红单端孢菌的致病机制奠定基础。以分离纯化后的粉红单端孢菌为试验材料,分别采用超声-TCA-丙酮、超声-磷酸-TCA-丙酮、超声-SDS、超声-TCA/丙酮-酚/SDS联合抽提和TCA/丙酮-酚/SDS联合抽提等5种方法提取菌体蛋白质,通过对比分析蛋白质含量以及纯度筛选出2种较好的提取方法。在筛选聚丙烯酰胺电泳凝胶浓度的基础上,再通过双向电泳对比分析,得出最佳的蛋白质提取方法。结果表明,12%的聚丙烯酰胺凝胶浓度获得的单向电泳图谱背景清晰且无严重的拖尾现象,超声-TCA/丙酮-酚/SDS联合抽提法获得蛋白样品的质量浓度和含量分别为6.650 mg/mL和2.993 mg/g,该法提取蛋白通过SDS-PAGE分析条带清晰,双向电泳分析可获得1 238个独立清晰的蛋白点。由此可知,超声-TCA/丙酮-酚/SDS联合抽提法获得的粉红单端孢菌体蛋白适合于双向电泳分析及蛋白质组学研究。
In order to optimize the protein extraction method suitable for two-dimensional electrophoresis of Trichothecium roseum and further study the pathogenic mechanism of T. roseum on the proteomic level,the isolated and purified T. roseum were chosen to extract the total protein by 5 methods:ultrasonic-TCA-acetone,ultrasonic-phosphoric acid-TCA acetone,ultrasonic-SDS,ultrasonic-TCA/acetone-phenol/SDS combined extraction and TCA/acetone-phenol/SDS combined extraction,respectively. Meanwhile,two better extraction methods were screened by comparative analysis of protein content and purity. Under the appropriate gel concentration of polyacrylamide electrophoresis,the best protein extraction method was obtained by two-dimensional electrophoresis. The results showed that 12% polyacrylamide gel concentration had clear background and no serious tailing. Moreover,the concentration and content of protein samples obtained by the method of ultrasonic-TCA/acetone-phenol/SDS combined extraction were 6.650 mg/mL and 2.993 mg/g respectively. Furthermore,clear bands were obtained in the extracted protein by SDS-PAGE analysis and 1 238 independent clear protein spots were obtained by two-dimensional electrophoresis analysis. It is suggested that the T. roseum protein obtained by the combined extraction method of ultrasonic-TCA/acetone-phenol/SDS was suitable for two-dimensional electrophoresis analysis and further proteomic research.
In order to optimize the protein extraction method suitable for two-dimensional electrophoresis of Trichothecium roseum and further study the pathogenic mechanism of T. roseum on the proteomic level,the isolated and purified T. roseum were chosen to extract the total protein by 5 methods:ultrasonic-TCA-acetone,ultrasonic-phosphoric acid-TCA acetone,ultrasonic-SDS,ultrasonic-TCA/acetone-phenol/SDS combined extraction and TCA/acetone-phenol/SDS combined extraction,respectively. Meanwhile,two better extraction methods were screened by comparative analysis of protein content and purity. Under the appropriate gel concentration of polyacrylamide electrophoresis,the best protein extraction method was obtained by two-dimensional electrophoresis. The results showed that 12% polyacrylamide gel concentration had clear background and no serious tailing. Moreover,the concentration and content of protein samples obtained by the method of ultrasonic-TCA/acetone-phenol/SDS combined extraction were 6.650 mg/mL and 2.993 mg/g respectively. Furthermore,clear bands were obtained in the extracted protein by SDS-PAGE analysis and 1 238 independent clear protein spots were obtained by two-dimensional electrophoresis analysis. It is suggested that the T. roseum protein obtained by the combined extraction method of ultrasonic-TCA/acetone-phenol/SDS was suitable for two-dimensional electrophoresis analysis and further proteomic research.
引文
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[10] 任淑艳,鲁淑荣.套袋苹果果实病虫害的发生与防治[J].河北果树,2007(3):12-13.doi:10.3969/j.issn.1006-9402.2007.03.007.Ren S Y,Lu S R.Occurrence and control of pests and diseases of bagged apple fruit[J].Hebei Fruits,2007(3):12-13.
[11] Niu L L,Bi Y,Bai X D,Zhang S G,Xue H L,Li Y C,Wang Y,Calderón-Urrea A.Damage to Trichothecium roseum caused by sodium silicate is independent from pH[J].Canadian Journal of Microbiology,2016,62(2):161-172.doi:10.1139/cjm-2015-0657.
[12] Hong C X,Michailides T J.Prune,plum,and nectarine as hosts of Trichothecium roseum in california orchards[J].Plant Disease,2007,81(1):112.
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