lncRNA AOANCR在蓝光信号调控红曲霉次级代谢中的作用研究

The biosynthesis pathways of the three polyketide secondary metabolites of Monascus are related to each other, so it is difficult to regulate them respectively, thus causing industrial problems such as frequent citrinin contamination in fermentation products, difficulties in obtaining high-purity single pigment component by fermentation and lower yield of Monacolin K. Long non-coding RNA (lncRNA) has a swift and flexible response in regulation and it is expected that each metabolite can be precisely regulated by using it as a target. Based on the previous studies, the applicant found that lncRNA AOANCR may regulate the secondary metabolism of Monascus by negative regulation of the alternating oxidase coding gene-mraox, but the specific mechanism is unknown. This study will first adopt FISH technique to locate subcellular localization of lncRNA AOANCR, and then use RNA interference or CRISPR/Cas9 technique for gene deletion to obtain mutons. Next, mutons and original strains are induced by blue light, and the differences of transcriptomes, metabolites and alternate respiration intensity between them before and after blue light induction are obtained. Finally, by conducting comprehensive comparison between all the differences of the two, its role in regulating the secondary metabolism of Monascus by blue light can be inferred. This study is deemed as a theoretical supplement to the regulation of the secondary metabolic mechanism of Monascus by blue light and its theoretical significance can provide theoretical support for the regulation with lncRNA as a target in production.

红曲霉三种聚酮次级代谢产物生物合成途径相关，难以单独调控，造成发酵产品中常有桔霉素污染；高纯度单一色素组分难以发酵获得；莫纳克林K产量偏低的产业难题。长链非编码RNA（lncRNA）调控反应迅速、灵活，以其为靶点有望对各代谢产物分别进行精准调控。申请人在前期研究发现，蓝光照射下lncRNA AOANCR可能通过负调节交替氧化酶的编码基因-mraox进而调控红曲霉次级代谢，但机制未知。本研究将首先采用FISH技术对lncRNA AOANCR细胞定位，用RNA干扰或CRISPR/Cas9 技术进行基因缺失，获得突变子。再对突变子和原始菌株分别蓝光诱导，得到二者在蓝光诱导前后的转录组、代谢产物和交替呼吸强度差异。最后将二者各项差异进行综合比较即可推断出其在蓝光调控红曲霉次级代谢中的作用。本研究是蓝光调控红曲霉次级代谢机制的理论补充，具有理论意义也为生产中以lncRNA为靶点进行调控提供理论支持。

红曲霉次级代谢产物包括：红曲色素、莫纳克林 K、桔霉素等。由于三者合成途径相关，造成发酵产品中常有桔霉素污染；高纯度单一色素组分难以发酵获得；MK 的产量偏低的产业难题。蓝光能够调控红曲霉桔霉素、M K 和色素合成。本人通过分析转录组数据发现：lncRNA可能在蓝光诱导条件下通过调控交替氧化酶编码基因(mraox)的表达进而调控次级代谢。lncRNA可能是精准调控红曲霉发酵的重要研究方向。我们分成了四个部分进行研究：1.从发酵学结果论证了lncRNA AOANCR和mraox调控红曲霉桔霉素代谢调控机理2.对pH5和7两个培养条件下的红曲霉转录组进行比较分析，在pH7的培养条件下找到204条lncRNA，在pH5的培养条件下找到205条lncRNA，并进一步进行分析。3. lncRNA AOANCR的功能研究工作。设计杂交探针，采用荧光原位杂交技术对lncRNA进行亚细胞定位，根据该lncRNA在细胞内的位置，推测其功能并选择 RNAi或CRISPR/Cas9 技术对lncRNA进行功能缺失，构建得到 lncRNA AOANCR功能缺失突变株。目前已经完成亚细胞定位，敲除工作正在进行。4.研究发现lncRNA AOANCR和mraox组合在pH诱导的外部环境调节中也存在代谢调控作用，进一步证明了lncRNA AOANCR可能是一个红曲霉代谢调控中的关键lncRNA。通过本项目研究，我们发现了lncRNA AOANCR和交替氧化酶编码基因(mraox)是红曲霉次级代谢调控关键因子。不仅光照能够诱导红曲霉lncRNA表达改变，pH条件的改变，也能诱导红曲霉lncRNA的改变，进而调控代谢。以前的红曲霉代谢研究停留在转录组层面，现在看来，研究能够调控mRNA的lncRNA对于深入研究丝状真菌的代谢调控有重要的学术意义，对于红曲霉工业化生产也具有重要的指导意义。