基于多组学技术研究5'-核苷酸酶介导3'-dAMP去磷酸化调控菌类中药虫草素生物合成的机制

Wild resources of fungal traditional Chinese medicine (FTCM) is becoming scarce, and its fermentation mycelium has become a good substitute. Cordycepin (3'-deoxyadenosine) is a key component of Cordyceps sinensis that plays an important biological activity, but its biosynthesis mechanism is not clear. Studies have shown that 5'-nucleotide (5'-NT) could mediate dephosphorylation of adenosine monophosphate (AMP) into adenosine in mammalian cells, but it has not been studied whether has a similar effect in FTCM. The preliminary study which based on multi-omics sequencing results of Hirsutella sinensis found that the increase of 5'-NT gene expression was positively correlated with the accumulation of cordycepin, and the results of fermentation control were further indicated that 5'-NT plays an positive role in the biosynthesis of cordycepin, whereas its functions and mechanism of action need to be studied urgently. We proposed cordycepin was synthesized by dephosphorylation of 3'-deoxygenation adenosine monophosphate (3'-dAMP) mediated by 5'-NT in fungus medicine, and 5'-NT plays a key role in the biosynthesis of cordycepin. This study intends to adopt the methods such as iTRAQ and gene editing, and arms to analyze and identify the functions of 5'-NT, and finally confirms the effect of 5'-NT on the biosynthesis of cordycepin by mediating the dephosphorylation of 3'-dAMP in FTCM. This study is expected to reveal a new mechanism of biosynthesis of cordycepin in FTCM, and provides more scientific basis for the metabolic regulation of cordycepin biosynthesis.

菌类中药野生资源日益匮乏，其发酵菌丝体成为了良好替代品。虫草素（3'-脱氧腺苷）是冬虫夏草发挥重要生物活性的关键成分，但其生物合成机制尚不明确。研究表明5'-核苷酸酶（5'-NT）在哺乳动物细胞中可介导腺苷一磷酸（AMP）去磷酸化生成腺苷，但在菌类中药中是否有相似作用尚未研究。前期冬虫夏草中国被毛孢转录组学研究发现，5'-NT基因表达上调与虫草素的积累正相关，发酵调控进一步表明5'-NT在虫草素生物合成中具有促进作用，但其功能及作用机制亟需研究。我们推测5'-NT可通过介导菌类中药3'-脱氧腺苷一磷酸（3'-dAMP）的去磷酸化合成虫草素，并在其生物合成中发挥关键作用。本研究拟采用iTRAQ和基因编辑等技术，分析和确定5'-NT的功能，明确5'-NT通过介导3'-dAMP去磷酸化调控虫草素的生物合成。本研究有望揭示菌类中药虫草素生物合成新机制，为代谢调控虫草素生物合成提供更充分的科学依据。

菌类中药野生资源日益匮乏，其发酵菌丝体成为了良好替代品。虫草素（cordycepin，3'-脱氧腺苷），是从菌类中药中分离得到的一种嘌呤生物碱或腺苷类似物，具有抗菌、抗肿瘤、免疫调节等多种生物学活性，但其生物合成机理尚不清楚。研究表明5'-核苷酸酶（5'-NT）在哺乳动物细胞中可介导腺苷一磷酸（AMP）去磷酸化生成腺苷，但在菌类中药中是否有相似作用尚未研究。本研究采用RNA-Seq转录组学测序分析、蛋白质组iTRAQ技术、异源表达及功能鉴定等方法，分析和确定了5'-NT的功能，明确了5'-NT通过介导3'-dAMP去磷酸化调控虫草素的生物合成。基于转录组学在转录组水平上研究了5'-NT与虫草素生物合成的相关性，共鉴定到2764个差异表达基因(DEGs)，其中上调1737个，下调1027个；结合qPCR和RNA-Seq基因表达数据预测腺苷生物合成途径表明，腺苷积累增加是ndk、ADK、APRT基因下调与AK、5'-NT基因上调共同作用的结果。接着，本研究采用一种高效的基因组挖掘方法从菌类中药中国被毛孢中挖掘5'-NT基因。鉴定并克隆了一个5'-NT基因，对其进行了生物信息学分析，并首次在毕赤酵母GS115中进行了异源表达。底物特异性分析表明，5'-NT对含有不同碱基的底物表现出不同的磷酸酶活性；圆二色谱测定结果表明，5'-NT在低温环境下对虫草素的生物合成具有重要作用。这些结果为人工控制虫草素的生物合成提供更科学的依据和新的思路。本研究探索了菌类中药虫草素的生物合成新机制，初步阐明了5'-NT在菌类中药虫草素生物合成中的作用新机制，也为人工调控虫草素的生物合成提供更充分的科学依据。

内容获取失败，请点击重试