Cepharanthine is a secondary metabolite isolated from Stephania. It has been reported that it has anti-conronaviruses activities including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we assemble three Stephania genomes (S. japonica, S. yunnanensis, and S. cepharantha), propose the cepharanthine biosynthetic pathway, and assess the antiviral potential of compounds involved in the pathway. Among the three genomes, S. japonica has a near telomere-to-telomere assembly with one remaining gap, and S. cepharantha and S. yunnanensis have chromosome-level assemblies. Following by biosynthetic gene mining and metabolomics analysis, we identify seven cepharanthine analogs that have broad-spectrum anti-coronavirus activities, including SARS-CoV-2, Guangxi pangolin-CoV (GX_P2V), swine acute diarrhoea syndrome coronavirus (SADS-CoV), and porcine epidemic diarrhea virus (PEDV). We also show that two other genera, Nelumbo and Thalictrum, can produce cepharanthine analogs, and thus have the potential for antiviral compound discovery. Results generated from this study could accelerate broad-spectrum anti-coronavirus drug discovery.

Mentha is a commonly used spice worldwide, which possesses medicinal properties and fragrance. These characteristics are conferred, at least partially, by essential oils such as menthol. In this study, a gap-free assembly with a genome size of 414.3 Mb and 31,251 coding genes was obtained for Mentha suaveolens ‘Variegata’. Based on its high heterozygosity (1.5%), two complete haplotypic assemblies were resolved, with genome sizes of 401.9 and 405.7 Mb, respectively. The telomeres and centromeres of each haplotype were almost fully annotated. In addition, we detected a total of 41,135 structural variations. Enrichment analysis demonstrated that genes involved in terpenoid biosynthesis were affected by these structural variations. Analysis of volatile metabolites showed that M. suaveolens mainly produces piperitenone oxide rather than menthol. We identified three genes in the M. suaveolens genome which encode isopiperitenone reductase (ISPR), a key rate-limiting enzyme in menthol biosynthesis. However, the transcription levels of ISPR were low. Given that other terpenoid biosynthesis genes were expressed, M. suaveolens ISPRs may account for the accumulation of piperitenone oxide in this species. The findings of this study may provide a valuable resource for improving the detection rate and accuracy of genetic variants, thereby enhancing our understanding of their impact on gene function and expression. Moreover, our haplotype-resolved gap-free genome assembly offers novel insights into molecular marker-assisted breeding of Mentha.

Artemisia annua is the only known plant source of the potent antimalarial artemisinin, which occurs as the low- and high-artemisinin producing (LAP and HAP) chemotypes. Nevertheless, the different mechanisms of artemisinin producing between these two chemotypes were still not fully understood. Here, we performed a comprehensive analysis of genome resequencing, metabolome, and transcriptome data to systematically compare the difference in the LAP chemotype JL and HAP chemotype HAN. Metabolites analysis revealed that 72.18% of sesquiterpenes was highly accumulated in HAN compared to JL. Integrated omics analysis found a DBR2-Like (DBR2L) gene may be involved in artemisinin biosynthesis. DBR2L was highly homologous with DBR2, belonged to ORR3 family, and had the DBR2 activity of catalyzing artemisinic aldehyde to dihydroartemisinic aldehyde. Genome resequencing and promoter cloning revealed that complicated variations existed in DBR2L promoters among different varieties of A. annua and were clustered into three variation types. The promoter activity of diverse variant types showed obvious differences. Furthermore, the core region (−625 to 0) of the DBR2L promoter was identified and candidate transcription factors involved in DBR2L regulation were screened. Thus, the result indicates that DBR2L is another key enzyme involved in artemisinin biosynthesis. The promoter variation in DBR2L affects its expression level, and thereby may result in the different yield of artemisinin in varieties of A. annua. It provides a novel insight into the mechanism of artemisinin-producing difference in LAP and HAP chemotypes of A. annua, and will assist in a high yield of artemisinin in A. annua.

For steady transgenic expression, lentiviral vector-mediated gene delivery is a commonly used technique. One question that needs to be explored is how external lentiviral vectors and overexpressed genes perturb cellular homeostasis, potentially altering transcriptional networks. In this study, two Human Embryonic Kidney 293T (HEK293T)-derived cell lines were established via lentiviral transduction, one overexpressing green fluorescent protein (GFP) and the other co-overexpressing GFP and ADORA3 following puromycin selection to ensure stable genomic integration. Genes with differentially transcript utilization (gDTUs) and differentially expressed genes (DEGs) across cell lines were identified after short-read and long-read RNA-seq. Only 31 genes were discovered to have changed in expression when GFP was expressed, although hundreds of genes showed variations in transcript use. In contrast, even when co-overexpression of GFP and ADORA3 alters the expression of more than 1000 genes, there are still less than 1000 gDTUs. Moreover, DEGs linked to ADORA3 overexpression play a major role in RNA splicing, whereas gDTUs are highly linked to a number of malignancies and the molecular mechanisms that underlie them. For the analysis of gene expression data from stable cell lines derived from HEK293T, our findings provide important insights into changes in gene expression and alternative splicing.

Phenotypic modifications and their effects on cellular functions through the up-regulation of target gene expression have frequently been observed in genetic studies, but the unique roles of cell lines and their introduced plasmids in influencing these functions have not been fully revealed. In this research, we developed two distinct cell lines derived from the A549 cell line: one that stably overexpresses GFP and another that is a polyclonal stable line overexpressing both GFP and P2RY12. We then utilized transcriptome sequencing (RNA-seq) technology to screen out differentially expressed genes (DEGs) and genes with differential transcript usage (gDTUs) after GFP overexpression (GFP-OE) and P2RY12 overexpression (P2RY12-OE). We found that, compared with A549, there were more than 1700 differentially expressed genes (DEGs) in both GFP-OE and P2RY12-OE cells, while only 866 DEGs were identified in GFP-OE and P2RY12-OE cells. Notably, the differences in transcript usage were relatively minor, with only over 400 genes exhibiting changes across all three groups. The functional analysis of DEGs and gDTUs showed that they were both highly enriched in the pathways associated with cell proliferation and migration. In summary, we performed an extensive analysis of the transcriptome profile of gene expression and alternative splicing with GFP-OE and P2RY12-OE, enhancing our comprehension of how genes function within cells and the processes that control gene expression.