曲霉菌中菌核形成相关转录因子SclR和EcdR对次生代谢产物的调控作用分析

Aspergillus species is widely distributed in grains, soil, and organisms, that is not only saprophyte which plays an important role in the decomposition of organic-matter in the environment, but also important eukaryote in fermentation and food processing industry. In our previous study, we have ever identified two novel predicted basic helix-loop-helix (bHLH) protein–encoding genes, sclR and ecdR in Aspergillus oryzae. As predicted bHLH transcription factors, they co-regulated the sclerotium formation through interaction with each other. However, the further function and mechanism are still unclear. In this study, by analyzing the DNA microarray (or RNA-Seq) and investigating the changes in secondary metabolite production between the gene disrupted strain/gene over-expressing strain and control strain, we will further illuminate the influence of the two transcription factors SclR and EcdR on secondary metabolites production and related-genes expression level on the whole genome. A. oryzae is speculated to be domesticated from the ancestor of A. flavus. One of the differences between these two Apergilli is that most strains of A. oryzae have lost the ability to form sclerotia or have an extremely low ability relative to A. flavus. At the same time, A. oryzae also lost the mycotoxin biosynthesis ability. Therefore, we consider that the sclerotium formation-related transcription factors also affect the mycotoxin biosynthesis in fungi such as A. flavus. According to the above results, for these genes which significantly affect the secondary metabolites production, we will further construct their gene disrupted strain and gene over-expressing strain in A. flavus. Through the investigation of changes in secondary metabolites production between the disrupted/over-expressing strain and control strain and the analysis of the toxicity, we will try to analyze the roles of sclerotium formation-related transcription factors in the biosynthesis of mycotoxins. Taken together, using the genome similarity and the difference of morphological characteristics such as sclerotium formation ability between A.oryzae and A. flavus, in this study, we aim to reveal the molecular mechanisms of transcription factors SclR and EcdR and other related factors in regulating the biosynthesis of secondary metabolites containing the mycotoxins in fungi.

曲霉菌广泛分布在谷物, 土壤和有机体上, 不仅是环境中对有机物分解起着重要作用的腐生菌, 也是发酵工业和食品加工业方面重要的真核微生物。申请人在对米曲霉的研究当中，新发现了两个basic helix-loop-helix (bHLH)转录因子SclR和EcdR,它们通过相互作用对菌核的形成有着重要的调控作用。在本研究中，申请人通过调查它们的基因敲除/过表达菌株当中次生代谢产物的变化，来研究这两个转录因子对次生代谢产物生产的影响。并结合DNA microarray分析结果，掌握它们对次生代谢产物生产的调控作用机制。利用黄曲霉与米曲霉之间的基因组的相似性和形态特征（菌核形成能力）的不同，研究黄曲霉中菌核形态和真菌毒素之间的关联性，并对其进行系统分析。希望通过本次菌核形成相关转录因子的进一步功能分析, 能够有效调控次生代谢产物的生产。为今后的畜牧业，医药业，和疾病研究领域方面的研究奠定理论基础。

曲霉菌广泛分布在包括谷物和土壤等各种自然环境中, 不仅对环境中有机物分解起着重要作用, 也是发酵工业和食品加工业方面重要的丝状真菌。申请人在本研究当中，对两个basic helix-loop-helix (bHLH)转录因子SclR和EcdR功能进行了探索，研究这两个转录因子对次生代谢产物生产的影响。研究表明，尽管这两个转录因子影响分生孢子的生产，但其中SclR转录因子还对菌株的生长，碳源利用，以及整个碳代谢都有着更重要的影响。研究表明，sclR基因敲除菌株在液体培养条件下表现了明显的生物量的减少以及胞内蛋白的降解。另外蛋白质组比较分析结果还建议SclR重要地参与包括糖解途径在内的各种碳代谢过程。尤其在其中的糖解过程中，sclR基因敲除导致三个限速酶的表达受到限制。由此可见，SclR是通过广域调控碳代谢过程，从而重要地影响各种次级代谢产物的生产。对sclR基因敲除菌株的包括次级代谢产物的代谢物进行分析，结果表明，一些有机酸，氨基酸和糖醇类化合物重要地减少了。尤其其中的一些有机酸是三羧酸循环的中间产物，说明SclR还重要地参与到包括三羧酸循环在内的能量代谢。本研究通过对转录因子SclR的功能分析, 有望为今后进一步研究碳代谢途径提供新的思路，以及为今后的曲霉属，尤其米曲霉在发酵产业以及医药业等领域的应用奠定理论基础。