Whole-transcriptome analysis and construction of ceRNA-miRNA-target gene regulatory networks in anther development of Chinese cabbage (Brassica campestris L. ssp. pekinensis) [Anther & mixed parts miRNA-seq]

Anther development is a complex process, and the study of its molecular mechanism has an important impact on plant breeding. This study aims to identify microRNA (miRNA), mRNA, long non-coding RNA (lncRNA), and circular RNA (circRNA) related to anther development of Chinese cabbage, so as to construct competitive endogenous RNA (ceRNA) regulatory networks and provide valuable knowledge for the exploration of pollen development mechanism of Chinese cabbage. A total of 9055 mRNA, 585 miRNA, 1344 lncRNA, and 165 circRNA were identified as differentially expressed in the anther of Chinese cabbage compared with Mix (roots, stems and leaves) by whole-transcriptome sequencing. The anther-related ceRNA-miRNA-target gene regulatory network through miRNA targeting relationships was constructed and 450 pairs of ceRNA relationships, including 97 DEmiRNA-DEmRNA, 281 DEmiRNA-DElncRNA, and 23 DEmiRNA-DEcircRNA interactions were obtained in Chinese cabbage. The genes in the ceRNA network were enriched in the pathways including starch and sucrose metabolism, carbon metabolism, pyruvate metabolism and carbon fixation in photosynthetic organisms, plant hormone signal transduction, and RNA degradation. This study identified some important genes and their interaction lncRNAs, circRNAs, and miRNAs involved in microsporogenesis (BraA06g035480.3C), tapetum and callose layer development (BraA09g009280.3C, BraA04g028920.3C, and BraA10g022680.3C etc), pollen wall formation (BraA06g000980.3C, BraA02g023130.3C, and BraA10g029650.3C etc), and anther dehiscence (BraA10g027200.3C, BraA04g023740.3C, and BraA04g030860.3C etc). Additionally, we analyzed the promoter activity of six anther predominant expression genes, and the results showed that they were all expressed specifically in the anther of Chinese cabbage. This study lay the foundation for further research on the molecular mechanism of anther growth and development. Overall design: we performed the whole-transcriptome sequencing of anther and Mix (roots, stems and leaves), and identified expression changes of miRNAs, lncRNAs, circRNAs, and mRNAs, and analyzed their potential regulatory roles with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG)

花药发育是一类复杂的生物学过程，对其分子机制的解析对于植物育种工作具有重要价值。本研究旨在鉴定与大白菜花药发育相关的微小RNA（microRNA, miRNA）、信使RNA（mRNA）、长链非编码RNA（long non-coding RNA, lncRNA）以及环状RNA（circular RNA, circRNA），以此构建竞争性内源RNA（competitive endogenous RNA, ceRNA）调控网络，为解析大白菜花粉发育的分子机制提供理论依据。本研究通过全转录组测序，在大白菜花药与混合样本（根、茎、叶）的比较中，共鉴定出9055个差异表达mRNA、585个差异表达miRNA、1344个差异表达lncRNA以及165个差异表达circRNA。本研究基于miRNA的靶向调控关系，构建了大白菜花药相关的ceRNA-miRNA-靶基因调控网络，共获得450对ceRNA调控关系，其中包含97个差异表达miRNA-差异表达mRNA、281个差异表达miRNA-差异表达lncRNA以及23个差异表达miRNA-差异表达circRNA的互作组合。该ceRNA网络中的基因显著富集于淀粉与蔗糖代谢、碳代谢、丙酮酸代谢、光合生物碳固定、植物激素信号转导以及RNA降解等通路。本研究还鉴定出多个参与关键生物学过程的重要基因及其互作的lncRNA、circRNA与miRNA：包括参与小孢子发生的基因（BraA06g035480.3C）、参与绒毡层与胼胝质层发育的基因（如BraA09g009280.3C、BraA04g028920.3C与BraA10g022680.3C等）、参与花粉壁形成的基因（如BraA06g000980.3C、BraA02g023130.3C与BraA10g029650.3C等）以及参与花药开裂的基因（如BraA10g027200.3C、BraA04g023740.3C与BraA04g030860.3C等）。此外，本研究对6个花药优势表达基因的启动子活性进行了分析，结果显示这些基因均在大白菜花药中特异性表达。本研究为后续解析花药生长发育的分子机制奠定了理论基础。整体实验设计：本研究对大白菜花药与混合样本（根、茎、叶）进行了全转录组测序，鉴定了miRNA、lncRNA、circRNA以及mRNA的表达差异，并通过基因本体论（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析探究了这些RNA的潜在调控功能。