转录因子INO4调控高山被孢霉ARA油脂合成的分子机制研究

Arachidonic acid (ARA) is an important polyunsaturated fatty acid in human body.It is high demand in Global, Mortierella alpinais an important microorganism in ARA industry.However, the domestic production cost is high, and the product quality is lower than the international standard, so it is difficult to develop the international market.Technological breakthroughs are urgently needed to improve the yield and quality of ARA oil in industrial production, which is a major basic application problem.Transcription factor acting as regulators of gene expression has pleiotropism.Based on the transcriptome data, INO4 the key transcription factors regulating ARA synthesis, were screened and cloned by bioinformatics analysis.The transcription was transferred into alpine spores due to INO4,and was overexpressed and knocked out by CRISPR/Cas9,and study the regulatory mechanism of ARA synthesis in Mortierella alpina.The functions of transcription factors and its regulatory pathway were revealed by using overexpression or RNA interference technology.The use of chromatin immunoprecipitation-depth sequencing and gel blocking technique were used to clarify the target genes of the transcription factors INO4 and their interactions. The ARA lipid synthesis network was revealed by regulation of transcription factors INO4. The results of the study will help improve ARA's industrial production level and product quality, enhance its international competitiveness, and lay a foundation for the breeding of other important polyunsaturated fatty acid oil producing strain and the optimization of fermentation conditions.

花生四烯酸（ARA）是人体重要的多不饱和脂肪酸，全球需求量大，高山被孢霉是ARA工业生产的重要微生物，但国内生产成本高，产品品质低于国际标准，难以开拓国际市场，亟需技术突破以提高工业生产ARA油脂产量及品质，是重大基础应用问题。转录因子作为基因表达调控子具有一因多效功能，本项目基于已有转录组数据，利用生物信息学分析筛选并克隆调控ARA合成的重要转录因子INO4，将重要转录因子INO4转入高山被孢霉超表达及CRISPR/Cas9敲除，结合转录组和脂质组学技术，深入解析其重要功能及脂质合成途径；利用染色质免疫共沉淀-深度测序和凝胶阻滞技术明确重要转录因INO4调控的靶基因及相互作用，从而解析转录因子INO4在转录前对ARA油脂合成网络的调控。研究结果有助于提升ARA工业生产水平和产品品质，增强国际竞争力，同时可为选育其他重要多不饱和脂肪酸油脂高产菌及发酵条件优化奠定基础。

二十二碳六烯酸（DHA）和花生四烯酸（ARA）是多不饱和脂肪酸的代表，具有重要生理功能，不仅可用于婴幼儿奶粉添加剂，且对糖尿病、心脑血管防治有重要作用，需求量日益增大。高山被孢霉是ARA油脂工业化生产菌株，解析转录因子INO4对ARA油脂合成调控分子机制，有助于提高ARA油脂工业生产水平和产品品质。通过本项目研究，取得以下研究成果：（1）高山被孢霉精细基因组测序及重要转录因子INO4基因的挖掘和克隆：利用高山被孢霉菌株基因组数据，通过两种不同的方法从基因组中共筛选到25个高山被孢霉候选bHLH转录因子，结合候选转录因子与真菌bHLH转录因子进化树分析，并与已报道INO4二级结构比较，最终筛选出5个候选转录因子INO4基因并进行克隆，分析上述基因序列理化特性，发现均定位于细胞核且为亲水性蛋白，不存在跨膜结构域，保守结构域α螺旋比较丰富，具有典型的bHLH结构；（2）高山被孢霉遗传转化体系的优化及包含转录因子INO4基因载体构建：建立并优化适用于本项目高山被孢霉的遗传转化体系，成功构建了以萎锈灵为筛选压力，包含转录因子INO4的pCAMBIA1303超表达载体和敲除载体；（3）MaINO4转录因子调控高山被孢霉ARA油脂合成的分子机制：MaINO4转录因子与脂质合成及脱饱和基因的表达密切相关，具有调控下游脂质合成代谢的功能，其基因表达量与高山被孢霉脂质中ARA含量，尤其是磷脂中ARA含量成正相关，揭示了MaINO4转录因子调控高山被孢霉ARA油脂合成的分子机制。上述研究成果为选育其他重要多不饱和脂肪酸油脂高产菌及发酵条件优化提供参考，且对于提升我国多不饱和脂肪油脂产业的国际竞争力具有重要意义。项目已发表高水平SCI论文2篇，已修回高水平SCI论文1篇，申请发明专利1项，参与编写教材1项。