大型真菌中谷氨酸生产菌的快速筛选

Rapid screening of glutamate-producing macrofungi

摘要:

【背景】大型真菌是重要的食品和药物资源，可产生包括必需氨基酸在内的多种营养成分。谷氨酸是食用大型真菌中呈现鲜味的重要功能分子之一，是衡量食用菌营养价值的一个重要指标。【目的】基于伞菌目中谷氨酸合酶同源基因的保守性，对大型真菌中含有谷氨酸的菌株资源进行筛选，为野生大型真菌资源的挖掘及谷氨酸产品的开发提供指导。【方法】通过对伞菌目中已报道的25株谷氨酸合酶基因序列进行比对分析，在谷氨酸合酶保守的外显子区域设计了2对简并引物GS Ag_les F1/R1和GS Ag_les F2/R2，并将其用于对伞菌目13个科的65株大型真菌进行PCR筛选。另外，还采用异硫氰酸苯酯衍生化的方法结合LC-MS和HPLC对这65个菌株产生的谷氨酸进行定量分析，进而评估该筛选方法的筛选效率。【结果】获得筛选引物的筛选效率为86.2%，并且引物GS Ag_les F1/R1的筛选效率明显优于GS Ag_les F2/R2。优化后的伞菌目大型真菌谷氨酸发酵培养条件为：LMM培养基，28℃、200 r/min，2—4 d。结合PCR和HPLC的分析结果评估该引物筛选的真阳性率为81.5%，应用范围涉及伞菌目的口蘑科、泡头菌科、伞菌科、丝膜菌科、粪伞科、离褶伞科、侧耳科、鬼伞科和鸟巢菌科9个科。【结论】本研究获得的筛选引物在伞菌目产生谷氨酸的菌株筛选中显示出了良好的应用前景，为谷氨酸生产菌株提供了高效、便捷的筛选技术方法。

关键词:谷氨酸;谷氨酸合酶;分子标记;食用菌;高效筛选

Abstract:

[Background] Macrofungi are important food and drug resources, which can produce a variety of nutrients including essential amino acids. Glutamate, one of the important functional molecules of edible macrofungi, presents umami taste and is an important index to measure the nutritional value of edible fungi. [Objective] We evaluated the resources of macrofungal strains containing glutamate based on the conserved homologous genes of glutamate synthase in Agaricales, aiming to guide the exploitation of wild macrofungal resources and the manufacture of glutamate products. [Methods] We compared the glutamate synthase gene sequences from 25 strains and analyzed the conserved exon region. Two pairs of degenerate primers, GS Ag_les F1/R1 and GS Ag_les F2/R2, were designed for the conserved exon region of glutamate synthase and used for PCR screening of 65 strains belonging to 13 families of Agaricales. In addition, phenylisothiocyanate derivatization was combined with LC-MS and HPLC to quantify the glutamate produced by the 65 strains, and then the screening efficiency of this method was evaluated. [Results] The screening efficiency of primers designed in this study was 86.2%, and the screening efficiency of the primer pair GS Ag_les F1/R1 was significantly better than that of GS Ag_les F2/R2. The fermentation conditions of Agaricales for the production of glutamate were optimized as follows:LMM medium, 28℃, 200 r/min and 2-4 days. The results of PCR and HPLC showed that the primer screening method in this study had a true positive rate of 81.5%, and the application scope involved multiple families including Tricholomataceae,Physalacriaceae, Agaricaceae, Cortinariaceae, Bolbitiaceae, Lyophyllaceae, Pleurotaceae, Psathyrellaceae and Nidulariaceae of Agaricales. [Conclusion] The primers obtained in this study exhibit a good application prospect in the screening of glutamate-producing strains of Agaricales.

Key words:glutamic acid;glutamic acid synthase;molecular marker;edible mushroom;efficient screening