红曲霉繁殖相关brlM基因鉴定及功能分析

Identification and function analysis of the brlM gene related to Monascus reproduction

摘要:

brlA作为曲霉分生孢子形成的中心调控路径中最上游的转录因子，能够诱导下游特异基因的表达调控分生孢子的产生，brlA缺失将导致曲霉无法产生分生孢子，同时生长代谢发生改变，但对不同菌种的影响有显著差异。【目的】探究brlA同源基因brlM在红曲霉中的基因功能，探索红曲霉繁殖调控机制。【方法】从紫色红曲霉Mp-21菌株中克隆了brlA同源基因brlM。利用同源重组原理，用农杆菌介导转化法构建了brlM基因缺失突变株(△brlM)，分析△brlM和野生菌株Mp-21在菌落表型、显微结构、生长速率和次生代谢产物等方面的差异，明确brlM基因在红曲霉中的主要功能。【结果】在形态水平上，brlM基因缺失菌株导致菌丝生长更加旺盛，赋予菌落更加蓬松的表型；显微观察发现△brlM失去了有性繁殖产生闭囊壳的能力，但却提升了无性繁殖产生分生孢子的能力；同时代谢产物红曲色素、莫纳可林K和桔霉素产量显著下降。【结论】红曲霉brlM基因的功能与曲霉属中的brlA并不相同，brlM基因在红曲霉有性繁殖中的作用不可替代。本研究的结果对进一步探索丝状真菌繁殖调控机制提供了新的思路。

Abstract:

brlA gene, as the initiator in the central regulatory pathway in Aspergillus conidiation, modulates the downstream conidiation-specific genes. Aspergillus without brlA fails to produce conidia, with growth and metabolism changed. However, the effect of brlA deletion is significantly different among different strains. [Objective] To explore the functions of brlM in Monascus, a homologous gene of brlA, and the regulatory mechanism of Monascus reproduction. [Methods] brlM was cloned from Monascus purpureus Mp-21. Based on homologous recombination, the brlM-deleted mutant (△brlM) was constructed by Agrobacterium tumefaciens-mediated transformation, and the colony phenotype, microstructure, growth rate, and secondary metabolites were compared between △brlM and the wild-type Mp-21 to clarify the functions of brlM in Monascus.[Results] For the morphology, △brlM showed more vigorous mycelial growth and a more fluffy phenotype of the colony than the wild type. According to the microscopy, △brlM lost the ability of producing cleistothecia through sexual reproduction but had stronger ability of produce conidia through asexual reproduction. Meanwhile, the yield of Monascus pigment, monacolin K, and citrinin of mutant △brlM was significantly lower than that of the wild type. [Conclusion] Different from brlA in Aspergillus, brlM gene is irreplaceable in the sexual reproduction of Monascus. This study provides a new idea for further research on the regulatory mechanism of the reproduction of filamentous fungi.