基于转录组测序的冬虫夏草虫草素生物合成研究

Study on bio-synthesis of cordycepin in Ophiocordyceps sinensis based on RNA-seq

目的对冬虫夏草进行转录组测序,为虫草素生物合成提供依据。方法利用Illumina/Solexa Hi Seq 2500高通量测序平台对冬虫夏草菌丝体和子实体转录组进行测序、数据组装,分析并预测虫草素生物合成途径、相关基因及差异表达量,发现代谢途径中多数酶在菌丝体发育阶段的表达水平较高。采用c DNA克隆技术从新鲜冬虫夏草子实体中克隆到核糖核苷酸还原酶(RNR)基因亚基。结果其中RNR是腺苷代谢的关键酶,从转录组数据中挖掘到RNR大、小亚基各1条,及4条RNR同源序列。RNR大亚基(RNRL)c DNA全长2 733 bp,编码910 aa,RNR小亚基(RNRM)c DNA全长1 257 bp,编码418 aa。通过保守区域和功能结构域分析发现主要催化活性位点位于RNRL,RNRM含有铁结合蛋白保守位点,大小亚基均含有二聚体和亚基结合区域。结论基于转录组测序预测,以RNR为切入点研究虫草素合成途径,为最终揭示虫草素生物合成机制提供数据支持。

Objective Recent years, some studies have been studied on the biosynthesis of cordycepin, but it is not clear. To sequence the transcriptomes of the Ophiocordyceps sinensis which could provide the basis for revealing the bio-synthesis mechanism of cordycepin. Methods In this study, by Illumina/Solexa Hi Seq 2500 technology, the transcriptomes of the O. sinensis fungus（anamorph） and the fruiting body（teleomorph） was sequenced, assembled and analyzed. By RT-PCR, the full lengths of RNRL（RNR large subunit） and RNRM（RNR small subunit） c DNA were cloned from the fresh O. sinensis fruit body. Results The pathway and the genes involved in cordycepin biosynthesis were predicted. Among of them, RNR was the critical enzyme in the metabolism of adenosine, also predicted to play an important role in the biosynthesis of cordycepin. From the transcriptome data, one large, one small subunits, and four similar sequences of RNR were found. RNRL m RNA was 2 733 bp, encoding 910 aa and RNRM m RNA 1 257 bp, encoding 418 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in RNRL, RNRM contained a ferritin-like conserved sequence. Conclusion This study would be established for revealing the bio-synthesis mechanism of cordycepin.