Data_Sheet_3_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.

水生苔藓植物门类中的肝叶苔（Radula marginata）作为一种具有潜力的类似大麻素化合物替代来源，然而，由于缺乏遗传信息，对这些类似大麻素化合物合成途径的分子机理的深入探究受到了阻碍。鉴于此，我们对R. marginata的转录组进行了深度测序、从头组装和注释，从而鉴定并验证了相关大麻素生物合成途径的基因。总计，我们鉴定出11,421个编码1,554种酶的潜在基因，涉及145个生物合成途径。值得注意的是，我们鉴定了包括其两个初级中间体——反式 stilbene 酸（SA）和香叶基焦磷酸（GPP）在内的，大麻二酚酸（CBGA）生物合成过程中的所有上游基因。我们利用定量实时PCR技术验证了所有这些基因的表达。我们对反式 stilbene 合成酶（STS）的蛋白质结构进行了表征，该酶在R. marginata中被认为与橄榄酸同源。此外，通过代谢组学方法，我们利用电喷雾离子化质谱（ESI-MS/MS）和气相色谱质谱（GC-MS）鉴定了CBGA类似化合物。转录组分析揭示了来自39个家族的1,085个转录因子（TF）。比较分析显示，在R. marginata中，六个转录因子家族被独特地预测。此外，生物信息学分析预测了大量简单序列重复（SSR）和非编码RNA（ncRNA）。我们的研究结果共同为类似大麻素化合物的潜在前体基因的生物合成提供了机理上的见解，并为R. marginata提供了一种新的转录组资源。本研究中生成的大规模转录组资源将进一步作为参考转录组，用于探究Radulaceae科。