Table13_Investigating nicotine pathway-related long non-coding RNAs in tobacco.XLSX

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with low or no protein-coding ability, which play essential roles in various biological processes in plants. Tobacco is an ideal model plant for studying nicotine biosynthesis and metabolism, and there is little research on lncRNAs in this field. Therefore, how to take advantage of the mature tobacco system to profoundly investigate the lncRNAs involved in the nicotine pathway is intriguing. By exploiting 549 public RNA-Seq datasets of tobacco, 30,212 lncRNA candidates were identified, including 24,084 large intervening non-coding RNAs (lincRNAs), 5,778 natural antisense transcripts (NATs) and 350 intronic non-coding RNAs (incRNAs). Compared with protein-coding genes, lncRNAs have distinct properties in terms of exon number, sequence length, A/U content, and tissue-specific expression pattern. lincRNAs showed an asymmetric evolutionary pattern, with a higher proportion (68.71%) expressed from the Nicotiana sylvestris (S) subgenome. We predicted the potential cis/trans-regulatory effects on protein-coding genes. One hundred four lncRNAs were detected as precursors of 30 known microRNA (miRNA) family members, and 110 lncRNAs were expected to be the potential endogenous target mimics for 39 miRNAs. By combining the results of weighted gene co-expression network analysis with the differentially expressed gene analysis of topping RNA-seq data, we constructed a sub-network containing eight lncRNAs and 25 nicotine-related coding genes. We confirmed that the expression of seven lncRNAs could be affected by MeJA treatment and may be controlled by the transcription factor NtMYC2 using a quantitative PCR assay and gene editing. The results suggested that lncRNAs are involved in the nicotine pathway. Our findings further deepened the understanding of the features and functions of lncRNAs and provided new candidates for regulating nicotine biosynthesis in tobacco.

长链非编码RNA（long non-coding RNAs, lncRNAs）是指长度超过200碱基对、编码蛋白质能力极低或完全丧失的转录本，在植物的各类生物学过程中发挥着关键作用。烟草是研究尼古丁生物合成与代谢的理想模式植物，但目前该领域内针对lncRNAs的研究仍较为匮乏。因此，如何依托成熟的烟草研究体系，深入探究参与尼古丁通路的lncRNAs，是一项极具研究意义的课题。本研究利用549组公开的烟草RNA测序（RNA-Seq）数据集，共鉴定得到30212个lncRNA候选分子，其中包含24084个基因间长链非编码RNA（large intervening non-coding RNAs, lincRNAs）、5778个天然反义转录本（natural antisense transcripts, NATs）以及350个内含子非编码RNA（intronic non-coding RNAs, incRNAs）。与蛋白质编码基因相比，lncRNAs在外显子数量、序列长度、A/U碱基含量以及组织特异性表达模式等维度均呈现出显著差异。lincRNAs表现出不对称的进化特征：来自林烟草（Nicotiana sylvestris, S）亚基因组的lincRNAs占比高达68.71%。我们预测了这些lncRNAs对蛋白质编码基因的潜在顺/反式调控效应。其中，104个lncRNAs被鉴定为30个已知microRNA（microRNA, miRNA）家族成员的前体，另有110个lncRNAs被预测为39个miRNAs的潜在内源靶模拟物。结合加权基因共表达网络分析结果与打顶样本RNA-seq数据的差异表达基因分析，我们构建了包含8个lncRNAs与25个尼古丁相关编码基因的共表达子网。通过定量PCR实验与基因编辑技术，我们证实7个lncRNAs的表达可被茉莉酸甲酯（MeJA）诱导，并可能受转录因子NtMYC2调控。本研究结果表明lncRNAs参与了尼古丁通路的调控过程，进一步加深了学界对lncRNAs特征与功能的认知，同时为烟草尼古丁生物合成的调控提供了全新的候选靶点。