黄曲霉野生型及LaeA缺陷型菌株胞外差异蛋白鉴定

Identification of extracellular differential proteins of Aspergillus flavus wild type and LaeA deficient strains

【目的】分析黄曲霉野生型产毒株及LaeA缺陷型无毒株在以小麦、玉米为培养基质中生长时的胞外差异蛋白,为进一步明确LaeA因子在黄曲霉产毒调控中的作用提供基础数据。【方法】将小麦及玉米籽粒粉碎后灭菌并加水配制成培养黄曲霉菌的基质,在产毒株(黄曲霉CA14野生型)及无毒株(LaeA缺陷型)培养0、48和72 h时取样,采用SDS-PAGE分别分析黄曲霉毒素B1(AFB1)及胞外差异蛋白,并采用液质联用四极杆飞行时间质谱技术[LC-MS-MS(Q-TOF)]对获得的胞外差异蛋白进行鉴定。【结果】黄曲霉野生型及LaeA缺陷型菌株均能将小麦和玉米基质中的小分子蛋白迅速转化,随着培养时间延长,LaeA缺陷型菌株的胞外蛋白较野生型菌株略少,不产生黄曲霉毒素。产毒株及无毒株胞外差异蛋白条带共有7个,主要为淀粉酶A、碱性蛋白酶、木聚糖酶F3和亮氨酸氨基肽酶A等生理酶类。【结论】黄曲霉产毒株与无毒株的胞外差异蛋白为生理酶类蛋白,该类酶蛋白主要与营养摄入有关,且影响黄曲霉菌丝的生长。

Objective]The present study was conducted to analyze extracellular differential proteins of Aspergillus flavus wild type and LaeA deficient strains cultured in wheat and corn substrates, in order to provide data for further understanding the roles of regulatory factor LaeA in regulation of aflatoxin production. [Method]The wheat and corn grains were grinded, sterilized and made into culture substrate by adding proper amount of water. Samples were taken at 0, 48 and 72 h after being inoculated with toxigenic strains（A. flavus CA14 wild type） and avirulent strains（LaeA deficient type） in substrates. SDS-PAGE was applied to analyze aflatoxin B1（AFB1） and extracellular differential proteins, and LC-MS-MS（Q-TOF） technology was employed to identify the extracellular differential proteins. [Result]A. flavus wild type and LaeA deficient strains could degrade small molecular proteins in wheat and corn substrates quickly. As time extended, LaeA deficient strains contained fewer extracellular differential proteins than wild type strains and produced no aflatoxin. There were seven extracellular differential protein bands in toxigenic strains and avirulent strains, which were mainly biological enzymes such as alpha-amylase A, alkaline protease, 1,4-beta-xylanase F3 and leucine aminopeptidase A.[Conclusion]The extracellular differential proteins in A. flavus toxigenic strains and avirulent strains belong to biological enzymes. These enzymes are related to nutrition intake and affect the growth of A. flavus mycelia.