适用于双向电泳分析的链格孢菌体蛋白提取方法的筛选
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摘要
为筛选出一套适用于双向电泳分析的链格孢菌蛋白的提取方法,以该菌为研究材料,首先采用TCA-丙酮法、磷酸-TCA-丙酮法、SDS提取法和TCA/丙酮-SDS/酚抽提法分别提取菌丝蛋白质,然后进行蛋白质浓度的测定和单向SDS-PAGE试验,并建立双向电泳体系。结果表明:TCA/丙酮-SDS/酚抽提法所获得的蛋白质浓度为15.9 mg/mL,分别是其他三种方法所得蛋白浓度的2.64、4.61和1.43倍;SDS-PAGE图谱中形成的条带数目最多、丰度最好;所获得的2-DE图谱背景清晰干净,无明显的横纵条纹,分辨率高,可辨别的蛋白质点数为1 138个,是TCA-丙酮法所分离蛋白点数的1.89倍。由此可知,TCA/丙酮-SDS/酚抽提法为链格孢菌蛋白提取的最优方法,该方法所提取的蛋白质适用于双向电泳分析及后续的蛋白质组学研究。
In order to screen the extraction methods of total protein from Alternaria alternata suitable for two-dimensional electrophoresis analysis, the mycelia proteins in the pathogen were extracted by four methods of TCA-acetone, phosphate-TCA-acetone, SDS and TCA/acetone-SDS/phenol, respectively. Furthermore, the protein concentration was analyzed and the separation effect of total protein was detected by SDS-PAGE.In addition, a suitable 2-D gel electrophoresis system for mycelia protein was established. The results showed that the protein concentration extracted by the method of TCA/acetone-SDS/phenol reached 15.9 mg/mL, which was 2.64, 4.61 and 1.43 times of that extracted by the methods of TCA-acetone, phosphate-TCA-acetone and SDS, respectively. Moreover, the most and clearest protein strips in SDS-PAGE gel were obtained by TCA/acetone-SDS/phenol extraction method, and the 2-DE map with a distinct background, high resolution, without obvious horizontal and vertical stripes were also obtained. The number of protein points reached 1 138, which is 1.89 times of that isolated by the TCA-acetone method. It is suggested that the TCA/acetone-SDS/phenol extraction method is the best extraction method of mycelia protein from Alternaria alternata. Meanwhile, the protein extracted by the method is suitable for two-dimensional electrophoresis analysis and subsequent proteomic research.
In order to screen the extraction methods of total protein from Alternaria alternata suitable for two-dimensional electrophoresis analysis, the mycelia proteins in the pathogen were extracted by four methods of TCA-acetone, phosphate-TCA-acetone, SDS and TCA/acetone-SDS/phenol, respectively. Furthermore, the protein concentration was analyzed and the separation effect of total protein was detected by SDS-PAGE.In addition, a suitable 2-D gel electrophoresis system for mycelia protein was established. The results showed that the protein concentration extracted by the method of TCA/acetone-SDS/phenol reached 15.9 mg/mL, which was 2.64, 4.61 and 1.43 times of that extracted by the methods of TCA-acetone, phosphate-TCA-acetone and SDS, respectively. Moreover, the most and clearest protein strips in SDS-PAGE gel were obtained by TCA/acetone-SDS/phenol extraction method, and the 2-DE map with a distinct background, high resolution, without obvious horizontal and vertical stripes were also obtained. The number of protein points reached 1 138, which is 1.89 times of that isolated by the TCA-acetone method. It is suggested that the TCA/acetone-SDS/phenol extraction method is the best extraction method of mycelia protein from Alternaria alternata. Meanwhile, the protein extracted by the method is suitable for two-dimensional electrophoresis analysis and subsequent proteomic research.
引文
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[23] 赵明治,杨秀芬,张明,等.一种促进植物根系生长的极细链格孢菌蛋白质分离、纯化和生物功能[J].中国生物防治,2007(2):170-173.
[24] DAMERVAL C,DE VIENNE D,ZIVY M,et al.Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins[J].Electrophoresis,2010,7(1):52-54.
[25] 张小泉.丙烷脒对灰葡萄孢作用的蛋白质组学初步研究[D].杨凌:西北农林科技大学,2009.
[26] WANG Wei,VIGNANI R,SCALI M,et al.A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis [J].Electrophoresis,2006,27:2782-2786.
[27] BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J].Analytical Biochemistry,1976,72(1/2):248-254.
[28] 陈晶瑜,郭宝峰,何付丽,等.适合双向电泳的植物全蛋白提取方法比较[J].中国农学通报,2010,26(23):97-100.
[29] WU Fangsheng,WANG Mengyun.Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues [J].Analytical Biochemistry,1984,139(1):100-103.
[30] LAKSHMAN D K,NATARAJAN S S,LAKSHMAN S,et al.Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani [J].Mycologia,2008,100(6):867-875.
[31] CARPENTIER S C,WITTERS E,LAUKENS K,et al.Preparation of protein extracts from recalcitrant plant tissues:An evaluation of different methods for two-dimensional gel electrophoresis analysis [J].Proteomics,2005,5:2497-2507.
[32] FERNáNDEZ-ACERO F J,COLBY T,HARZEN A,et al.Proteomic analysis of the phytopathogenic fungus Botrytis cinerea during cellulose degradation [J].Proteomics,2009,9(10):2892-2902.
[33] 冯浩,王海英,冯雅琼,等.苹果树腐烂病菌菌丝蛋白双向电泳体系的建立[J].西北农林科技大学学报(自然科学版),2017,45(6):155-162.
[34] 刘原志,章强强.iTRAQ技术在真菌研究中的应用进展[J].中国真菌学杂志,2015,10(3):185-189.
[35] 谢秀枝,王欣,刘丽华,等.iTRAQ技术及其在蛋白质组学中的应用[J].中国生物化学与分子生物学报,2011,27(7):616-621.
[2] 张天宇.中国真菌志.第十六卷,链格孢属[M].北京:科学出版社,2003.
[3] YAGO J I,LIN C H,CHUNG K R.The SLT2 mitogen-activated protein kinase-mediated signalling pathway governs conidiation,morphogenesis,fungal virulence and production of toxin and melanin in the tangerine pathotype of Alternaria alternata[J].Molecular Plant Pathology,2011,12(7):653-665.
[4] 李庆亮,李捷,李夏鸣,等.细胞壁降解酶在苹果霉心病菌致病过程中的作用研究[J].中国农学通报,2015,31(31):90-95.
[5] 蔺经,杨青松,李晓刚,等.嘧霉胺对梨黑斑病菌(Alternaria alternata)的毒力及其药效评价[J].植物保护,2009,35(4):162-163.
[6] 黄颖倩.草莓病原菌及其拮抗菌的筛选、鉴定与抑菌特性初探[D].泰安:山东农业大学,2016.
[7] 梅秀凤.柑橘褐斑病和黑腐病病原及内生型链孢菌的比较研究[D].杭州:浙江大学,2016.
[8] 袁惠君,李虎军,贾鸿震,等.永登枸杞鲜果晾晒过程中霉腐病原真菌的分离鉴定[J].食品工业科技,2016,37(21):135-138.
[9] 刘瑜,王海,王艳丹,等.枸杞鲜果霉变菌种分离鉴定及其生物学特性[J].农业工程学报,2017,33(S1):374-380.
[10] 谢东锋,毕阳,邓建军,等.采前壳聚糖对厚皮甜瓜果实潜伏侵染及其采后主要病害的控制[J].甘肃农业大学学报,2008(2):96-99.
[11] 吴仁锋,汪志红.番茄早疫病研究概述[J].中国植保导刊,2009,29(3):16-18.
[12] 王婧,翟伟卜,高环,等.链格孢引起的病害严重危害农作物生产并危及农产品安全[J].植物保护,2017,43(4):9-15.
[13] 刘娟.柑桔园链格孢菌的鉴定与防治[D].重庆:西南大学,2001.
[14] HYON G S,IKEDA K,HOSOGI N,et al.Inhibitory effects of antioxidant reagent in reactive oxygen species generation and penetration of appressoria of Alternaria alternata Japanese pear pathotype [J].Phytopathology,2010,100(9):840-847.
[15] VéL?Z H,GLROOK N J,DAUB M E,et al.Mannitol biosynthesis is required for plant pathogenicity by Alternaria alternata[J].FEMS Microbiology Letters,2008,285(1):122-129.
[16] 苏玉斌.金龟子绿僵菌致病力与发育的差异蛋白质组学研究[D].福州:福建农林大学,2012.
[17] BHADAURIA V,ZHAO Wensheng,WANG Lixia,et al.Advances in fungal proteomics[J].Microbiological Research,2007,167:193-200.
[18] CARPENTIER S C,WITTERS E,LAUKENS K,et al.Preparation of protein extracts from recalcitrant plant tissues:An evaluation of different methods for two-dimensional gel electrophoresis analysis [J].Proteomics,2005,5(10):2497-2507.
[19] 江珊,周晓罡,苏源,等.氮胁迫条件下稻瘟病菌菌丝体蛋白质组学研究[J].扬州大学学报(农业与生命科学版),2012,33(4):72-76.
[20] FERNáNDEZ-ACERO F J,JORGE I,CALVO E,et al.Two-dimensional electrophoresis protein profile of the phytopathogenic fungus Botrytis cinerea[J].Proteomics,2006,6(S1):S88-S96.
[21] GONZáLEZ F R,ALORIA K,VALERO G J,et al.Proteomic analysis of mycelium and secretome of different Botrytis cinerea wild-type strains [J].Journal of Proteomics,2014,97:195.
[22] 舒灿伟,陈健仪,赵美,等.一种优化的适用于双向电泳的水稻纹枯病菌蛋白质提取方法[J].华中农业大学学报,2017,36(5):15-19.
[23] 赵明治,杨秀芬,张明,等.一种促进植物根系生长的极细链格孢菌蛋白质分离、纯化和生物功能[J].中国生物防治,2007(2):170-173.
[24] DAMERVAL C,DE VIENNE D,ZIVY M,et al.Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins[J].Electrophoresis,2010,7(1):52-54.
[25] 张小泉.丙烷脒对灰葡萄孢作用的蛋白质组学初步研究[D].杨凌:西北农林科技大学,2009.
[26] WANG Wei,VIGNANI R,SCALI M,et al.A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis [J].Electrophoresis,2006,27:2782-2786.
[27] BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J].Analytical Biochemistry,1976,72(1/2):248-254.
[28] 陈晶瑜,郭宝峰,何付丽,等.适合双向电泳的植物全蛋白提取方法比较[J].中国农学通报,2010,26(23):97-100.
[29] WU Fangsheng,WANG Mengyun.Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues [J].Analytical Biochemistry,1984,139(1):100-103.
[30] LAKSHMAN D K,NATARAJAN S S,LAKSHMAN S,et al.Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani [J].Mycologia,2008,100(6):867-875.
[31] CARPENTIER S C,WITTERS E,LAUKENS K,et al.Preparation of protein extracts from recalcitrant plant tissues:An evaluation of different methods for two-dimensional gel electrophoresis analysis [J].Proteomics,2005,5:2497-2507.
[32] FERNáNDEZ-ACERO F J,COLBY T,HARZEN A,et al.Proteomic analysis of the phytopathogenic fungus Botrytis cinerea during cellulose degradation [J].Proteomics,2009,9(10):2892-2902.
[33] 冯浩,王海英,冯雅琼,等.苹果树腐烂病菌菌丝蛋白双向电泳体系的建立[J].西北农林科技大学学报(自然科学版),2017,45(6):155-162.
[34] 刘原志,章强强.iTRAQ技术在真菌研究中的应用进展[J].中国真菌学杂志,2015,10(3):185-189.
[35] 谢秀枝,王欣,刘丽华,等.iTRAQ技术及其在蛋白质组学中的应用[J].中国生物化学与分子生物学报,2011,27(7):616-621.