基于组学研究红曲菌分生孢子产生的核心调控机制

Monascus spp. are famous for their fermentation product called red mold rice, which has been widely used in food coloring and blood lipid regulation. Conidia are the important propagules of Monascus spp., but their conidia are less yielded and productivity is far lower than that of Aspergillus spp., which largely affect the competitiveness of Monascus spp. and lead to easy contamination by other microorganisms in red mold rice fermentation. The comparative genomics analysis indicated that among all the sequenced Eurotiales fungi, except the central regulatory pathway of Monascus conidiation is in brlA-wetA model, others are in brlA-abaA-wetA model. Monascus genomes lack abaA regulatory gene and three structural genes for differentiation of sterigmata, in accordance with the fact that Monascus pycnidia lacks sterigmata structure. It suggests that Monascus genome lost these four genes during evolution, leading to the distinctive pycnidia structure against other Eurotiales fungi. In this study, these four genes will be inserted and expressed in M. ruber M7 by molecular biology techniques, to analyze the changes of pycnidia structure, conidia productivity, and production of related secondary metabolites. Meanwhile, transcriptomics techniques will be applied to analyze the brlA-wetA regulatory pathway of Monascus conidiation. In short, the findings in this study will not only provide guidance to improve Monascus conidia productivity, but also supplement to regulatory mechanism of fungal conidiation.

红曲菌，又称红曲霉，其发酵产物（红曲）广泛用于食品着色和调理血脂等。分生孢子是其重要繁殖体，但产量少，产率远低于曲霉等，这在很大程度上影响了红曲菌竞争力，导致红曲生产中容易被杂菌污染。基因组比较发现，在目前已测序的散囊目真菌中，除红曲菌分生孢子产生的核心调控通路为brlA-wetA外，其它均为brlA-abaA-wetA；红曲菌缺少调控小梗分化的调节基因abaA，并缺少与小梗分化相关的3个结构基因。显微观察发现，红曲菌的分生孢子器也正好缺少小梗。由此推测，红曲菌有可能是在进化过程“丢失”了相关基因。为此，本研究拟采用分子生物学手段将以上“丢失”的基因“回补”到红色红曲菌M7基因组中，分析“回补”前后分生孢子器结构、分生孢子产率以及与相关次生代谢产物的变化。同时，采用转录组学等分析红曲菌brlA-wetA产孢调控机制。研究结果不仅为提高红曲菌分生孢子产率提供指导，也可丰富真菌产孢调控机制。

红曲菌，又称红曲霉，其发酵产物红曲广泛用于食品着色和调理血脂等。分生孢子是其重要繁殖体。基因组比较发现，在目前已测序的散囊目真菌中，除红曲菌分生孢子产生的核心调控通路为brlA-wetA外，其它均为brlA-abaA-wetA；红曲菌缺少调控小梗分化的调节基因abaA。显微观察发现，红曲菌的分生孢子器也正好缺少小梗。由此推测，红曲菌有可能是在进化过程“丢失”了相关基因。为此，本研究采用分子生物学手段将来自构巢曲霉的abaA基因在红曲菌中进行表达，同时构建了红曲菌brlA和wetA敲除菌株。分析发现，与出发菌株相比，abaA表达菌株及brlA和wetA敲除菌株的菌落形态无明显差异。分生孢子形态进行比较发现，brlA和wetA敲除菌株分生孢子与出发菌株一样，单向串生于孢子梗囊泡的顶端，但abaA表达菌株中有少部分的分生孢子双向或者三向串生于孢子梗顶端。abaA表达基因可以促进红曲菌分生孢子产率，而wetA和brlA敲除菌株的产量下降。abaA表达菌株和brlA敲除菌株分子孢子萌发率及耐受力上升，而wetA敲除菌株分生孢子萌发率和耐受力下降。综上，abaA基因未能将红曲菌恢复成曲霉产孢模式，产孢核心调控基因wetA和brlA基因的敲除也没有体现出跟模式真菌构巢曲霉类似的现象。这些结果表明曲霉经典的产孢调控模式在红曲菌中并不适用，也表明红曲菌中有一套新的调控机制，这丰富目前的产孢认识。