Integrated miRNAome, transcriptome, and degradome analyses reveal the role of miRNA-mRNA modules in the biosynthesis of oridonin in Isodon rubescens

Abstract

Introduction: Isodon rubescens contains many bioactive diterpenoids, especially oridonin, which are used both as medicines and drinks. However, the structure and content of the diterpenoids in I. rubescens vary greatly in response to different ecological environments. MicroRNAs (miRNAs) play a pivotal role in the biosynthesis of secondary metabolites; but their roles in I. rubescens are obscure.

Methods: This research involved conducting miRNAome, transcriptome, and degradome sequencing analysis of three ecotypes of I. rubescens. Furthermore, the regulation of two candidate miRNA-mRNA modules was validated through a dual-luciferase reporter system.

Results: In this study, a total of 1634 miRNAs were identified from 9 miRNAome libraries of three I. rubescens ecotypes, which contained various contents of oridonin, lasiodonin, and rosthorin A. Furthermore, 99 DEMs and 8180 DEGs were obtained across three I. rubescens ecotypes, and the expressions of selected DEMs and DEGs were verified via reverse transcription quantitative PCR (RT-qPCR). A total of 8928 miRNA-mRNAs networks were identified by degradome analysis, and 23 miRNA-mRNA modules were enriched in the terpenoid biosynthesis pathway. Additionally, 92 negatively correlated DEM‒DEG modules were identified through integrated miRNAome, transcriptome, and degradome analyses, ath-miR858b_1ss21GA‒MYB and ath-miR408-3p_L-1R+1‒CYP72A219 modules were likely involved in oridonin biosynthesis in I. rubescens. Furthermore, the negative regulation of ath-miR858b_1ss21GA targeted MYB was validated through a dual-luciferase reporter system.

Discussion: This study revealed that Ath-miR858b_1ss21GA could repress MYB transcription, potentially downregulating the specific genes involved in the biosynthesis of oridonin and reducing oridonin accumulation in I. rubescens.

Keywords: Isodon rubescens; dual-luciferase reporter assay; miR858b_1ss21GA-MYB module; multiomics analysis; oridonin biosynthesis.

Figure 1

Phenotypic characteristics and analysis of the contents of five components in three Isodon rubescens ecotypes. (A) Phenotypic characteristics of three I. rubescens ecotypes. (B) HPLC chromatogram of the mixed reference solution and three I.rubescens ecotype samples. Numbers 1–5 represent rutin, lasiodonin, rosthorin A, rosmarinic acid, and oridonin, respectively. (C) Contents of the five components of the three I.rubescens ecotypes. All data are expressed as the mean standard deviations (x ± SD, n = 3). Error bars indicate SD. One-way ANOVA and the post-hoc test were used for multiple testing using SPSS 22.0. A box plot with Tukey’s significance markers (A–C) was used to illustrate the group differences. p < 0.01.

Figure 2

Length distributions of the total reads and unique microRNAs (miRNAs) in Isodon rubescens. (A) Length distribution of the total reads. (B) Number of unique miRNAs of different lengths.

Figure 3

First-nucleotide bias analysis of the microRNAs (miRNAs) in Isodon rubescens. (A–C) First-nucleotide percentages in total miRNAs (A), known miRNAs (B), and novel miRNAs (C).

Figure 4

Identification of the differentially expressed miRNAs (DEMs) and genes (DEGs) in three ecotypes of Isodon rubescens. (A) Identification of miRNAs expressed in three I. rubescens ecotypes. (B, C) Identification of the DEMs (B) and DEGs (C) in three I. rubescens ecotypes. (D) Expression heatmap of the DEMs identified in both HOr vs. NOr and LOr vs. NOr. The heatmap was generated by the standardized log2(TPM + 0.01). Red represents a high expression level, while blue represents a low expression level. HOr, from the Taihang Mountains; NOr, from the Funiu Mountains; LOr, from the Qinling Mountains.

Figure 5

Putative differentially expressed genes (DEGs) involved in oridonin biosynthesis in Isodon rubescens. The differential expression heatmap of the DEGs was generated by the standardized log2(average(FPKM) + 0.01). Red represents a high expression level, while blue represents a low expression level.

Figure 6

RT-qPCR analysis of the selected differentially expressed microRNAs (DEMs) and genes (DEGs). (A, B) Relative expression and transcription levels of the DEMs (A) and DEGs (B) in Isodon rubescens. Three biological replicates were used for each sample, and each biological replicate included three repeated techniques. Relative expression was analyzed with the 2^{−ΔΔ C t} method using U6 or the Actin gene as the internal reference for miRNA or mRNA, respectively.

Figure 7

Number analysis of the targets and microRNAs (miRNAs). (A) Target numbers of the top 15 miRNAs based on degradome analysis. (B) Number of the top 15 miRNA targets based on degradome analysis, where NA represents an undefined protein.

Figure 8

Top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in three comparison groups based on degradome transcripts. (A) Top 20 KEGG pathways in the HOr vs. NOr group. (B) Top 20 KEGG pathways in the LOr vs. NOr group. (C) The top 20 KEGG pathways in the HOr vs. LOr group. HOr, produced in the Taihang Mountains; NOr, produced in the Funiu Mountains; LOr, produced in the Qinling Mountains.

Figure 9

Putative microRNA (miRNA)–messenger RNA (mRNA) modules involved in oridonin biosynthesis in Isodon rubescens based on three degradome transcripts. The differential expression heatmaps of the miRNAs and target mRNAs were generated by the standardized log2(average(TPM) + 0.01) or log2(average(FPKM) + 0.01). Red represents high expression levels, while blue represents low expression levels.

Figure 10

Expression trends and nucleotide cleavage sites of the microRNAs (miRNAs) and negative targets in Isodon rubescens. (A) Expression trends of the miRNAs and negative targets in I. rubescens based on quantitative real-time PCR (qRT-PCR) analysis. (B) Target plots (T-plots) of the miRNA targets confirmed by degradome sequencing. In the T-plots, red dots indicate the miRNA-directed cleaved transcripts. The X-axis indicates the nucleotide position in the target complementary DNA (cDNA). The Y-axis indicates the number of reads of cleaved transcripts detected in the degradome cDNA library.

Figure 11

Relative LUC/REN ratios in ath-miR858b_1ss21GA+MYB and ath-miR408-3p_L-1R+1+CYP72A219-like.

Figure 12

Multi-omics analysis uncovers the microRNA (miRNA)–messenger RNA (mRNA) networks that regulate oridonin biosynthesis in Isodon rubescens.