摘要
目的对红曲霉中真菌毒素桔霉素进行定量分析,并研究桔霉素合成相关基因簇,为从基因水平上对桔霉素的产生进行调控、提高红曲霉相关食品的安全性提供理论依据。方法采用高效液相色谱(HPLC)法对3株红曲霉(紫色红曲霉YY-1、M2,丛毛红曲霉FJ-1)在液态发酵过程中产生的桔霉素开展定量分析,采用二代测序技术鉴定桔霉素合成基因簇的序列特征,应用实时定量聚合酶链式反应(RT-PCR)法进行相关基因表达水平分析。结果在固态培养和液态发酵过程中,3株红曲霉菌体生长情况无明显差异;在液态发酵过程中,HPLC结果显示M2桔霉素产量最高,其次为YY-1,FJ-1产量最少;M2、YY-1、FJ-1桔霉素合成基因簇相似度达99.9%;膜转运蛋白基因(orf5)、聚酮合酶基因(pksCT)、氧化还原酶基因(ctnB)、转录调节蛋白基因(ctnA)、脱氢酶基因(orf1、ctnE)六个基因表达水平在M2中最高,在FJ-1中最低。结论桔霉素的产量差异与桔霉素合成基因簇所表达酶的种类无关,而与其基因表达的调控相关。
Objective In order to regulate the production of citrinin from the gene level and to provide a theoretical basis for improving the safety of Monascus used in food production, the quantitative analysis of citrinin in three strains of Monascus was performed, and the related gene clusters were studied. Methods The quantitative analysis of citrinin in three strains of Monascus during liquid fermentation was analyzed by high performance liquid chromatography(HPLC). The citrinin gene clusters were sequenced at Novogene Corporation(Beijing, China) and expression levels of genes involved in citrinin gene cluster were analyzed by real time polymerase chain reaction(RT-PCR). Results There was no significant difference in the growth of Monascus purpureus YY-1, M2 and Monascus pilosus FJ-1 in both solid-state culture and liquid fermentation. In the liquid fermentation process, the yield of citrinin in M2 was the highest, followed by YY-1, and the production of citrinin in FJ-1 was the lowest. The similarity of the citrinin synthesis gene clusters in YY-1, M2 and FJ-1 reached 99.9%, exhibiting high conservation on gene level. The expression levels of six genes including membrane transport protein gene(orf5), polyketide synthetase gene(pksCT), oxidoreductase gene(ctnB), transcriptional regulation protein gene(ctnA) and dehydrogenase genes(orf1, ctnE) in the gene cluster were the highest in M2 and the lowest in FJ-1. Conclusion The citrinin yield was not related to the gene composition of citrinin synthesis gene cluster, but related to gene expression levels.
引文
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