适用于双向电泳分析的链格孢菌体蛋白提取方法的筛选
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SDSPAGE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;SDSPAGE图谱中形成的条带数目最多、丰度最好;所获得的2DE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。
关键词 链格孢; 采后病害; 蛋白质提取; 双向电泳; TCA/丙酮SDS/酚抽提法
中图分类号: S 432.1
文献标识码: ADOI: 10.16688/j.zwbh.2018285
Abstract In order to screen the extraction methods of total protein from Alternaria alternata suitable for twodimensional electrophoresis analysis, the mycelia proteins in the pathogen were extracted by four methods of TCAacetone, phosphateTCAacetone, SDS and TCA/acetoneSDS/phenol, respectively. Furthermore, the protein concentration was analyzed and the separation effect of total protein was detected by SDSPAGE.In addition, a suitable 2D gel electrophoresis system for mycelia protein was established. The results showed that the protein concentration extracted by the method of TCA/acetoneSDS/phenol reached 15.9 mg/mL, which was 2.64, 4.61 and 1.43 times of that extracted by the methods of TCAacetone, phosphateTCAacetone and SDS, respectively. Moreover, the most and clearest protein strips in SDSPAGE gel were obtained by TCA/acetoneSDS/phenol extraction method, and the 2DE 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 TCAacetone method. It is suggested that the TCA/acetoneSDS/phenol extraction method is the best extraction method of mycelia protein from Alternaria alternata. Meanwhile, the protein extracted by the method is suitable for twodimensional electrophoresis analysis and subsequent proteomic research.
Key words Alternaria alternata; postharvest disease; protein extraction; twodimensional electrophoresis; TCA/acetoneSDS/phenol method
鏈格孢Alternaria alternata是一种能侵染多种农作物和经济作物的重要植物病原真菌,广泛分布在土壤、空气和农作物残体中,可寄生、腐生和兼性寄生或腐生[1]。A.alternata是半知菌亚门链格孢属的模式种[2],其菌落边缘整齐,初白色,而后逐渐变为不同程度的褐色,气生菌丝呈短绒状,其分生孢子梗为深色,单枝,顶端单生或串生分生孢子,分生孢子呈黑褐色,形状大小不一,呈倒棍棒状或倒梨形,有横隔膜[3]。该菌可侵染苹果[4]、梨[5]、草莓[6]、柑橘[7]、枸杞[89]、甜瓜[10]、番茄[11]等多种果蔬,引起采后烂损,并且其侵染过程产生的交链孢霉毒素给果蔬及其制品销售带来食品安全隐患[12]。全面解析病菌的分子致病机理对制定该病害的有效控制策略有重要意义。目前,已有研究人员对该病菌分子致病机理开展了较为广泛的研究,但研究主要集中在毒素[13]和代谢产物[1415]上,从蛋白质水平揭示链格孢分子致病机理的研究尚未见报道。
近年来,蛋白质组技术已经广泛应用于植物病原真菌致病机理的研究中。双向电泳(twodimensional gel electrophoresis, 2DE)技术是蛋白质组学研究中一种有效的蛋白质分离方法,而获得高分辨率2DE图谱的关键步骤是蛋白质提取[16]。目前,适用于2DE技术的植物病原真菌总蛋白提取常用TCA/丙酮法[17]、酚抽提法[18]及其衍生的改良方法。江珊等[19]采用改进的TCA/丙酮法对稻瘟病菌Magnaporthe oryzae菌丝体蛋白质进行双向电泳分析,获得了高分辨率电泳图谱,较改进前TCA/丙酮法蛋白质点数量平均提高687个点。FernándezAcero等[20]研究了磷酸盐缓冲液预处理对TCA丙酮法提取灰葡萄孢Botrytis cinerea菌丝体总蛋白的效果,发现在TCA/丙酮沉淀蛋白质样品之前使用磷酸盐缓冲液进行预处理可显著提高灰葡萄孢菌丝体总蛋白的提取效果。González等[21]研究发现TCA/丙酮结合苯酚提取法所提取的灰葡萄孢菌丝体蛋白含量高于磷酸TCA丙酮法,且该方法提高了蛋白点的分辨率,减少了拖尾,有更多的蛋白点被检测出。舒灿伟等[22]对采用不同蛋白提取方法所提取的水稻纹枯病菌Rhizoctonia solani蛋白质样品质量进行比较研究,结果表明,优化的TCA/丙酮酚/SDS联合抽提法适用于进行双向电泳的水稻纹枯病菌蛋白质的提取。但目前有关链格孢中蛋白质的有效提取方法研究鲜有报道。
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