面团酸度和氮含量对扣囊复膜酵母孢子生长的影响研究

Effects of dough acidity and nitrogen content on spore growth of Saccharomycopsis fibuligera

目的探究面团培养基的酸度与氮含量对扣囊复膜酵母(Saccharomycopsis fibuligera)孢子生长分子机制的影响。方法采用面团培养基,在不同酸度与氮含量条件下培养扣囊复膜酵母孢子,通过荧光染色法观测孢子的活力;二甲氧唑黄[2.3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]-2H-tetrazolium hydroxide,XTT]比色法测定扣囊复膜酵母孢子生物被膜的生长动力学;实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)分析扣囊复膜酵母孢子的pH信号通路、Ras/cAMP/PKA信号通路和细胞周期关键基因的表达情况。结果面团培养基在pH 7.0时,扣囊复膜酵母孢子凋亡率为0.4%,活跃率为98.4%,pH信号通路中RIM101基因表达量最高,菌丝生长关键基因EFG1表达量最低;面团培养基在氮含量4.8%时,扣囊复膜酵母孢子凋亡率为0.8%,活跃率为96.4%,菌丝生长关键基因RAS1表达量较高。结论面团pH 7.0或氮含量4.8%时,扣囊复膜酵母孢子活力高,生物被膜形成能力强,扣囊复膜酵母孢子生长机制受细胞周期、pH信号调节通路、Ras/cAMP/PKA信号通路关键基因的调控。本研究可为扣囊复膜酵母孢子的工业化制备提供理论依据。

Objective To explore the effects of acidity and nitrogen content of dough medium on the molecular mechanism of spore growth of Saccharomycopsis fibuligera.Methods The spores of Saccharomycopsis fibuligera were cultured in dough medium under different acidity and nitrogen content,and the vitality of spores was recorded by fluorescence staining.The growth kinetics of biofilm was determined by 2.3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]-2H-tetrazolium hydroxide(XTT) colorimetric method.The expression levels of key genes related to the pH signaling pathway,Ras/cAMP/PKA signaling pathway and cell cycle were analyzed in Saccharomycopsis fibuligera spores by real-time fluorescence quantitative polymerase chain reaction(PCR).Results When the dough medium was at pH 7.0,the apoptosis rate of Saccharomycopsis fibuligera spore was 0.4%,and the active rate was 98.4%.The expression of RIM101 gene in the pH signaling pathway was the highest,while the expression of EFG1,a key gene for mycelial growth,was the lowest.When the nitrogen content of dough medium was 4.8%,the apoptosis rate of Saccharomycopsis fibuligera spores was 0.8%,the activity rate was 96.4%,and the expression of RAS1,a key gene for mycelial growth,was higher.Conclusion When the dough medium pH is 7.0 or the nitrogen content is 4.8%,the Saccharomycopsis fibuligera spore activity and biofilm formation ability are high.The growth mechanism of Saccharomycopsis fibuligera spores is regulated by the key genes of the cell cycle,pH signaling pathway and Ras/cAMP/PKA signaling pathway.This study can provide a theoretical basis for the industrial preparation of Saccharomycopsis fibuligera spores.