Jiang Mei, Chen Haimei, Du Qing, Wang Liqiang, Liu Xinyue, Liu Chang
School of Pharmaceutical Sciences, Qilu University of Technology (Shandong Academy of Sciences), Jinan, China.
Key Laboratory for Applied Technology of Sophisticated Analytical Instruments of Shandong Province, Shandong Analysis and Test Center, Qilu University of Technology (Shandong Academy of Sciences), Jinan, China.
Front Genet. 2021 Nov 5;12:645115. doi: 10.3389/fgene.2021.645115. eCollection 2021.
Circular RNAs (circRNAs) play various roles in cellular functions. However, no studies have been reported on the potential involvement of circRNAs in the biosynthesis of secondary metabolites in plants. Here, we performed a genome-wide discovery of circRNAs from root, stem and leaf samples of using RNA-Seq. We predicted a total of 2,476 circRNAs with at least two junction reads using circRNA_finder and CIRI, of which 2,096, 151 and 229 were exonic, intronic and intergenic circRNAs, respectively. Sequence similarity analysis showed that 294 out of 2,476 circRNAs were conserved amongst multiple plants. Of the 55 predicted circRNAs, 31 (56%) were validated successfully by PCR and Sanger sequencing using convergent and divergent primer pairs. Alternative circularisation analysis showed that most parental genes produced two circRNAs. Functional enrichment analyses of the parental genes showed that the primary metabolism pathways were significantly enriched, particularly the carbon metabolism. Differential expression analysis showed that the expression profiles of circRNAs were tissue-specific. Co-expression analysis showed 275 circRNAs, and their parental genes had significantly positive correlations. However, 14 had significantly negative correlations. Weighted gene co-expression network analysis showed that nine circRNAs were co-expressed with four modules of protein-coding genes. Next, we found 416 exonic circRNAs with miRNA-binding sites, suggesting possible interactions between circRNAs and miRNAs. Lastly, we found six validated circRNAs, namely, SMscf2473-46693-46978, SMscf3091-29256-29724, SMscf16-111773-112193, SMscf432-13232-13866, SMscf7007-10563-10888 and SMscf1730-1749-2013, which were originated from the genes involved in the biosynthesis of secondary metabolites. Their parental genes were acetyl-CoA C-acetyltransferase 1 (SmAACT1), 1-deoxy-d-xylulose-5-phosphate synthase 2 (SmDXS2), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1 (SmHDR1), kaurene synthase-like 2 (SmKSL2), DWF4 and CYP88A3, respectively. In particular, the correlation coefficient of SMscf2473-46693-46978 and SmDXS2 gene was 0.86 ( = 0.003), indicating a potential interaction between this pair of circRNA and its parent gene. Our results provided the first comprehensive catalogue of circRNAs in and identified one circRNA that might play important roles in the biosynthesis of secondary metabolites.
环状RNA（circRNAs）在细胞功能中发挥着多种作用。然而，尚未有关于circRNAs在植物次生代谢物生物合成中潜在作用的研究报道。在此，我们利用RNA测序对[植物名称]的根、茎和叶样本进行了全基因组环状RNA的发现。我们使用circRNA_finder和CIRI预测了总共2476个至少有两个连接读数的circRNAs，其中2096个、151个和229个分别为外显子circRNAs、内含子circRNAs和基因间circRNAs。序列相似性分析表明，2476个circRNAs中有294个在多种植物中保守。在55个预测的circRNAs中，31个（56%）通过使用收敛和发散引物对的PCR和桑格测序成功验证。可变环化分析表明，大多数亲本基因产生两种circRNAs。亲本基因的功能富集分析表明，初级代谢途径显著富集，尤其是碳代谢。差异表达分析表明，circRNAs的表达谱具有组织特异性。共表达分析表明，275个circRNAs与其亲本基因具有显著正相关。然而，有14个具有显著负相关。加权基因共表达网络分析表明，9个circRNAs与四个蛋白质编码基因模块共表达。接下来，我们发现416个具有miRNA结合位点的外显子circRNAs，表明circRNAs与miRNAs之间可能存在相互作用。最后，我们发现6个经过验证的circRNAs，即SMscf2473 - 46693 - 46978、SMscf3091 - 29256 - 29724、SMscf16 - 111773 - 112193、SMscf432 - 13232 - 13866、SMscf7007 - 10563 - 10888和SMscf1730 - 1749 - 2013，它们来源于参与次生代谢物生物合成的基因。它们的亲本基因分别是乙酰辅酶A C - 乙酰转移酶1（SmAACT1）、1 - 脱氧 - D - 木酮糖 - 5 - 磷酸合酶2（SmDXS2）、4 - 羟基 - 3 - 甲基丁 - 2 - 烯基二磷酸还原酶1（SmHDR1）、贝壳杉烯合酶样2（SmKSL2）、DWF4和CYP88A3。特别是，SMscf2473 - 46693 - 46978与SmDXS2基因的相关系数为0.86（P = 0.003），表明这对circRNA与其亲本基因之间存在潜在相互作用。我们的结果提供了[植物名称]中circRNAs的首个综合目录，并鉴定出一个可能在次生代谢物生物合成中发挥重要作用的circRNA。
