Whole-transcriptome sequencing reveals a vernalization-related ceRNA regulatory network in chinese cabbage (Brassica campestris L. ssp. pekinensis)

Abstract

Background: The transition from vegetative growth to reproductive growth involves various pathways. Vernalization is a crucial process for floral organ formation and regulation of flowering time that is widely utilized in plant breeding. In this study, we aimed to identify the global landscape of mRNAs, microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) related to vernalization in Chinese cabbage. These data were then used to construct a competitive endogenous RNA (ceRNA) network that provides valuable information to better understand the vernalization response.

Results: In this study, seeds sampled from the Chinese cabbage doubled haploid (DH) line 'FT' with or without vernalization treatment were used for whole-transcriptome sequencing. A total of 2702 differentially expressed (DE) mRNAs, 151 DE lncRNAs, 16 DE circRNAs, and 233 DE miRNAs were identified in the vernalization-treated seeds. Various transcription factors, such as WRKY, MYB, NAC, bHLH, MADS-box, zinc finger protein CONSTANS-like gene, and B3 domain protein, and regulatory proteins that play important roles in the vernalization pathway were identified. Additionally, we constructed a vernalization-related ceRNA-miRNA-target gene network and obtained 199 pairs of ceRNA relationships, including 108 DEmiRNA‒DEmRNA, 67 DEmiRNA‒DElncRNA, and 12 DEmiRNA‒DEcircRNA interactions, in Chinese cabbage. Furthermore, several important vernalization-related genes and their interacting lncRNAs, circRNAs, and miRNAs, which are involved in the regulation of flowering time, floral organ formation, bolting, and flowering, were identified.

Conclusions: Our results reveal the potential mRNA and non-coding RNAs involved in vernalization, providing a foundation for further studies on the molecular mechanisms underlying vernalization in Chinese cabbage.

Keywords: Chinese cabbage; Non-coding RNA; Vernalization; Whole transcriptome; ceRNA.

Fig. 1

Germinated seeds (A) and plants (B) of Chinese cabbage exposed to non-vernalized (Nor) and vernalized (Ver; 4 °C for 25 d) conditions

Fig. 2

Identification and analysis of mRNAs under vernalization. A Venn diagram showing the number of mRNAs in non-vernalized (Nor) and vernalized (Ver) Chinese cabbage seeds; (B) statistical analysis of the number of up- and downregulated differentially expressed mRNAs (DEmRNAs) identified between the Nor and Ver samples; (C) heat map; (D) Gene Ontology (GO) classifications; (E) GO enrichment analysis; and (F) Kyoto Encyclopedia of Genes and Genome (KEGG) pathway assignments for all of the DEmRNAs

Fig. 3

Identification and analysis of long-coding RNAs (lncRNAs) under vernalization. A Venn diagram showing the number of lncRNAs in non-vernalized (Nor) and vernalized (Ver) Chinese cabbage seeds; (B) statistics for the number of up- and downregulated differentially expressed lncRNAs (DElncRNAs) identified between the Nor and Ver samples; (C) heat map; (D) Gene Ontology (GO) classifications; (E) GO enrichment; and (F) Kyoto Encyclopedia of Genes and Genome (KEGG) pathway assignments for all DElncRNAs

Fig. 4

Comparison of the DElncRNA and DEmRNA structural characteristics and expression levels. A Transcript length distribution of DElncRNAs and DEmRNAs; (B) exon number of DElncRNAs and DEmRNAs; (C and D) ORF length distribution of DElncRNAs and DEmRNAs; and (E) DElncRNA and DEmRNA expression levels. DElncRNAs: differentially expressed long-coding RNAs; DEmRNAs: differentially expressed mRNAs

Fig. 5

Co-expression network of differentially expressed long-coding RNAs (DElncRNAs) and differentially expressed mRNAs (DEmRNAs)

Fig. 6

Identification and analysis of circular RNAs (circRNAs) under vernalization. A Venn diagram showing the number of circRNAs in non-vernalized (Nor) and vernalized (Ver) Chinese cabbage seeds; (B) statistics for the number of up- and downregulated differentially expressed circRNAs (DEcircRNAs) identified between the Nor and Ver samples; (C) heat map; (D) Gene Ontology (GO) classifications; (E) GO enrichment; and (F) Kyoto Encyclopedia of Genes and Genome (KEGG) pathway assignments for all DEcircRNAs

Fig. 7

Identification and analysis of microRNAs (miRNAs) under vernalization. A Venn diagram showing the number of miRNAs in non-vernalized (Nor) and vernalized (Ver) Chinese cabbage seeds; (B) statistics for the number of up- and downregulated differentially expressed miRNAs (DEmiRNAs) identified between the Nor and Ver samples; (C) heat map; (D) Gene Ontology (GO) classifications; (E) GO enrichment; and (F), Kyoto Encyclopedia of Genes and Genome (KEGG) pathway assignments for all DEmiRNAs

Fig. 8

Competitive endogenous RNA (ceRNA) network constructed with all differentially expressed mRNAs (DEmRNAs), differentially expressed long-coding RNAs (DElncRNAs), differentially expressed circular RNAs (DEcircRNAs), and differentially expressed microRNAs (DEmiRNAs)

Fig. 9

Quantitative real-time polymerase chain reaction (qRT‒PCR) analysis. qRT-PCR results for (A) differentially expressed mRNAs (DEmRNAs); (B) differentially expressed long-coding RNAs (DElncRNAs); (C) differentially expressed circular RNAs (DEcircRNAs); and (D) differentially expressed microRNAs (DEmiRNAs) in non-vernalized (Nor) and vernalized (Ver) Chinese cabbage seeds. **P < 0.01 and *P < 0.05, based on Duncan’s test

Fig. 10

Quantitative reverse transcriptase-polymerase chain reaction (qRT‒PCR) analysis. qRT-PCR results for the (A) differentially expressed mRNAs (DEmRNAs); (B) differentially expressed long-coding RNAs (DElncRNAs); (C), differentially expressed circular RNAs (DEcircRNAs); and (D) differentially expressed microRNAs (DEmiRNAs) in Chinese cabbage seeds treated with different vernalization times. Different letters indicate significant differences among the different treatments according to the least significant difference (LSD) test at P < 0.05