基于CRISPR/Cas9技术研究LncRNA靶向IDI基因调控茯苓新酸类化合物形成的分子机制

Poricoic acids are the main bioactive components in traditional Chinese medicine Poria cocos, but its content needs to be increased. Aiming at revealing the biosynthesisi of poricoic acids, various samples of Poria cocos at different poricoic acids contents were collected and transcriptomic analysis was carried out in our previous study.3 LncRNA genes that target-collocated to the key enzyme IDI gene were found. If biosynthesis of poricoic acids is regulated by LncRNA targeting to IDI gene? In this project, we plan to build target LncRNA editing mutant of Poria cocos with LncRNA knockout by CRISPR/Cas9 technology, to find out the correlation between target LncRNA, IDI gene and poricoic acids content, to verify the localization of LncRNA and analyze its binding point and interacting biomolecules, to investigate the regulatory mechanism of LncRNA on IDI gene expression and poricoic acids synthesis. This study will help to reveal the biosynthesis mechanism of poricoic acids and provide information for further strain improvement by target gene editing.

茯苓新酸类化合物是我国大宗药材茯苓的主要活性成分之一，但其含量有待提高以改善药用价值。明晰茯苓新酸类化合物形成调控机制是靶向提高其含量的前提，但目前该机制尚不清晰。申请人前期采集了不同茯苓样本，发现MVA通路中IDI是茯苓新酸类化合物形成关键酶，并筛选获得与IDI基因共定位的3条关键LncRNA基因。推测LncRNA可能通过靶向调控IDI基因转录，从而调控茯苓新酸类化合物形成。为验证该假说，本项目拟采用CRISPR/Cas9基因编辑技术对筛选的目标LncRNA基因进行敲除，分析LncRNA基因敲除茯苓中的LncRNA与IDI基因转录和茯苓新酸类化合物形成的相关性，分析目标LncRNA的细胞定位，探讨目标LncRNA的结合位点和互作分子，研究调控机制，为茯苓定向遗传育种提供基础。

茯苓是我国大宗药材，茯苓新酸类三萜物质是其主要活性成分之一，但其含量有待提高以改善药用价值。针对茯苓新酸生物合成调控机制尚不清晰，本项目采集了茯苓新酸存在差异的不同茯苓样本，转录组测序发现，MVA通路中IDI是茯苓新酸生物合成关键酶。通过LncRNA特性筛选构建了茯苓LncRNA数据集，并克隆获得与IDI基因共定位共表达的关键LncRNA基因。通过ATMT农杆菌侵染法和PEG介导原生质体法对比，优化了CRISPR/Cas9载体转化茯苓细胞的实验方法，采用CRISPR/Cas9基因编辑技术对筛选的目标LncRNA基因进行了靶向敲除。同时采用HPLC-Q-TOF-MS技术建立了茯苓样本中茯苓新酸类三萜物质成分的研究方法。对目标LncRNA的亚细胞定位进行了分析，初步探讨了目标LncRNA的可能调控作用机制。本项目成功构建了茯苓CRISPR/Cas9基因编辑体系，探讨了茯苓新酸类三萜物质成分的生物合成和调控，为茯苓优质种质资源培育和靶向遗传育种提供了实验基础。