Identification and functional analysis of ovarian lncRNAs during different egg laying periods in Taihe Black-Bone Chickens

Abstract

Introduction: Long non-coding RNA (lncRNA) refers to a category of non-coding RNA molecules exceeding 200 nucleotides in length, which exerts a regulatory role in the context of ovarian development. There is a paucity of research examining the involvement of lncRNA in the regulation of ovary development in Taihe Black-Bone Chickens. In order to further investigate the egg laying regulation mechanisms of Taihe Black-Bone Chickens at different periods, transcriptome analysis was conducted on the ovarian tissues at different laying periods. Methods: This study randomly selected ovarian tissues from 12 chickens for RNA-seq. Four chickens were selected for each period, including the early laying period (102 days, Pre), the peak laying period (203 days, Peak), and the late laying period (394 days, Late). Based on our previous study of mRNA expression profiles in the same ovarian tissue, we identified three differentially expressed lncRNAs (DE lncRNAs) at different periods and searched for their cis- and trans-target genes to draw an lncRNA-mRNA network. Results and discussion: In three groups of ovarian tissues, we identified 136 DE lncRNAs, with 8 showing specific expression during the early laying period, 10 showing specific expression during the peak laying period, and 4 showing specific expression during the late laying period. The lncRNA-mRNA network revealed 16 pairs of lncRNA-target genes associated with 7 DE lncRNAs, and these 14 target genes were involved in the regulation of reproductive traits. Furthermore, these reproductive-related target genes were primarily associated with signaling pathways related to follicle and ovary development in Taihe Black-Bone Chickens, including cytokine-cytokine receptor interaction, TGF-beta signaling pathway, tyrosine metabolism, ECM-receptor interaction, focal adhesion, neuroactive ligand-receptor interaction, and cell adhesion molecules (CAMs). This study offers valuable insights for a comprehensive understanding of the influence of lncRNAs on poultry reproductive traits.

Keywords: RNA-seq; Taihe Black-Bone Chicken; egg production; long non-coding RNA; ovary.

FIGURE 1

Characteristics of lncRNAs identified in the ovaries of Taihe Black-Bone Chickens. (A) Distribution of lncRNA type; (B) lncRNA and mRNA transcripts length; (C) lncRNA and mRNA exon number; (D) lncRNA and mRNA ORF length.

FIGURE 2

Comparison of expression level and the differential expression of lncRNAs in three groups. (A) The volcano plot of lncRNAs in Pre vs. Peak; (B) the volcano plot of lncRNAs in Pre vs. Late; (C) The volcano plot of lncRNAs in Late vs. Peak. (D) Venn diagram of DE lncRNAs of the three groups.

FIGURE 3

GO annotation of DE lncRNAs’ cis target genes for the top 30 items in (A) Pre vs. Peak, (B) Pre vs. Late, and (C) Late vs. Peak. GO annotation of DE lncRNAs’ trans target genes for the top 30 items in (D) Pre vs. Peak, (E) Pre vs. Late, and (F) Late vs. Peak.

FIGURE 4

The 20 most significant KEGG pathways of DE lncRNAs’ cis target genes in (A) Pre vs. Peak, (B) Pre vs. Late, and (C) Late vs. Peak. The 20 most significant KEGG pathways of DE lncRNAs’ trans target genes in (D) Pre vs. Peak, (E) Pre vs. Late, and (F) Late vs. Peak.

FIGURE 5

The lncRNA-mRNA interaction network depicting mRNAs regulated by specially expressed lncRNAs with specific expression during various periods. (A) mRNAs regulated in trans, (B) mRNAs regulated in cis, (C) mRNAs related to ovarian development. The triangles and inverted triangle exhibit upregulated and downregulated lncRNAs: red exhibits in the early laying period; purple exhibits during the peak laying period; blue exhibits during the late laying period. The green ellipses exhibit target genes.

FIGURE 6

Protein–protein interaction network of mRNAs regulated by lncRNAs specifically expressed across various periods. (A) The early laying period, (B) the peak laying period.

FIGURE 7

Validation of six differentially expressed lncRNAs and six differentially expressed mRNAs was performed by qPCR. The letters on the column diagram indicate significant (p < 0.05) differences at different stages. (A–F) represents lncRNA, and (G–L) represents mRNA.

FIGURE 8

A schematic diagram of the regulatory role of identified lncRNAs in ovarian function.