Data_Sheet_5_Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in Radula marginata.XLSX

The liverwort Radula marginata belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to do deep sequencing, de novo assembly and annotation of R. marginata transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1,554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA), including its two first intermediates, stilbene acid (SA) and geranyl diphosphate (GPP). Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS), which is considered as a homolog of olivetolic acid in R. marginata. Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS) and gas chromatography mass spectrometry (GC-MS). Transcriptomic analysis revealed 1085 transcription factors (TF) from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in R. marginata. In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs) and non-coding RNAs (ncRNAs). Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for R. marginata. The large-scale transcriptomic resource generated in this study would further serve as a reference transcriptome to explore the Radulaceae family.

肝叶苔Radula marginata隶属于苔藓植物门，是潜在的类似大麻素化合物的新来源。然而，由于缺乏遗传信息，对指导这些类似大麻素化合物合成的分子途径的机制性认识受到了阻碍。这促使我们对R. marginata转录组进行深度测序、从头组装和注释，从而鉴定和验证了大麻素生物合成途径的基因。总计，我们鉴定了编码来自145个生物合成途径的1,554个酶的11,421个假基因。有趣的是，我们鉴定了包括其两个初级中间体，即反式-二氢愈创木酸（SA）和香叶基焦磷酸（GPP）在内的，大麻二酚酸（CBGA）生物合成的上游所有基因。使用定量实时PCR对这些基因的表达进行了验证。我们已鉴定了 stilbene synthase（STS）的蛋白质结构，其在R. marginata中被认为是橄榄酸的同源物。此外，代谢组学方法使我们能够通过电喷雾电离质谱（ESI-MS/MS）和气相色谱质谱（GC-MS）鉴定CBGA类似化合物。转录组分析揭示了来自39个家族的1085个转录因子（TF）。比较分析表明，在R. marginata中有六个TF家族被独特地预测。此外，生物信息学分析预测了大量的简单序列重复（SSRs）和非编码RNA（ncRNAs）。我们的结果共同提供了对拟前体基因生物合成类似大麻素化合物的机制性见解，并为R. marginata提供了一个新的转录组资源。本研究生成的庞大转录组资源将进一步作为参考转录组，以探索Radulaceae科。