Data_Sheet_4_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.

苔藓植物门中的肝瓣苔（Radula marginata）作为一种潜在的类似大麻素化合物的替代来源，引起了广泛关注。然而，由于遗传信息的匮乏，对这些类似大麻素化合物合成途径的分子机制研究受到了阻碍。鉴于此，我们进行了肝瓣苔转录组的深度测序、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，我们鉴定了编码来自145个生物合成途径中的1,554种酶的11,421个潜在基因。值得注意的是，我们鉴定了包括其两个初级中间体——反式肉桂酸（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。所有这些基因的表达均通过实时荧光定量PCR进行了验证。我们对反式肉桂酸合成酶（STS）的蛋白质结构进行了表征，该酶在肝瓣苔中与橄榄酸具有同源性。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）识别了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）。比较分析表明，在肝瓣苔中，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揭示了类似大麻素化合物潜在前体基因的生物合成机制，并为肝瓣苔提供了一个新的转录组资源。本研究中生成的大规模转录组资源将作为参考转录组，用于进一步探索肝瓣苔科。