The candidate ssc-miR-221-3p and TYRP1 genes related to melanin synthesis were screened out based on transcriptome sequencing.
In this study, 17 significantly differentially expressed miRNAs and 1230 significantly differentially expressed genes were detected by transcriptome sequencing of black and white skin tissues of Jianbai Xiang Pig (Supplementary Table S1). The results showed that 10 miRNAs were up-regulated and 7 miRNAs were down-regulated, 738 differentially expressed genes were up-regulated and 492 were down-regulated (Fig. 1). According to GO and KEGG analysis, 9 miRNA target genes and 15 differentially expressed mRNAs were enriched in the melanin synthesis and tyrosine metabolism pathways (Supplementary Fig S1, Supplementary Table S2).
6 differentially expressed miRNAs (ssc-miR-181b, ssc-miR-196a, ssc-miR-221-3p, ssc-miR-320, unconservative-e_3_403490, unconservative_1_52628) and 9 differentially expressed mRNAs (SLC24A5, SLC45A2, SLC7A11, SLC7A5, MLANA, TYR, TYRP1, DCT, PMEL) were validated by qRT-PCR. The results showed that 6 miRNAs and 9 genes showed a pattern of expression changes between black and white skin tissues and a trend consistent with the transcriptome sequencing results (Fig. 2A-D), indicating that the data based on transcriptome sequencing in this study were accurate and reliable.
Figure 1 Transcriptome analysis of miRNA and mRNA in the black and white skin tissue of Xiang pigs. (A, B) miRNA and mRNA differential expression volcano map. (C, D) miRNA and mRNA differential expression clustering map. Columns represent different samples, rows represent different miRNAs and mRNAs, clustered by log10 (TPM + 1) values, red indicates high expression miRNAs and mRNAs, green indicates low expression miRNAs and mRNAs. S03, S04, S05; L13, L14, L15 are skin samples from the black coat on the back of Jianbai Xiang pigs, S02, S06, S07; L12, L16, L17 are skin samples from the white coat on the head of Jianbai Xiang pigs.
It was predicted using the RNA22 v2 online software that ssc-miR-221-3p could bind to the CDS region of the TYRP1 gene and regulate its transcription. Further analysis of the significantly differentially expressed miRNAs and mRNAs revealed that 17 miRNAs were significantly differentially expressed in Jianbai Xiang Pig's black and white skin tissues, and 92 target genes of 11 miRNAs were associated with differential genes (Fig. 2E), and 8 miRNAs(unconservative_3_403490,ssc-miR-10a-5p,ssc-miR-181b,ssc-miR-196a,ssc-miR-196b-5p,ssc-miR-221-3p,ssc-miR-320,ssc-miR-615) were upregulated in white skin tissues compared to black, and 3 miRNAs (ssc-miR-143-3p, ssc-miR-7143-3p, unconservative_X_1843986) were down-regulated in white skin tissues. Further analysis of 92 differential target genes revealed that only ssc-miR-221-3p targeting the TYRP1 gene was enriched to the melanin synthesis pathway, indicating that miRNA-221-3p could be a candidate miRNA for regulating melanin synthesis.
Figure 2 Experiments and correlation analysis. (A-D) Expression of selected miRNAs and genes in the black and white skin of the Xiang pig (**, P < 0.001). A is the TPM value of each miRNA by transcriptome sequencing, B is the mRNA expression of each gene detected by qRT-PCR, C is the FPKM value of each gene by transcriptome sequencing, and D is the qRT-PCR detection of each gene. mRNA expression. (E) Differential target genes and miRNAs enriched in melanin synthesis pathway and tyrosine metabolism notification. Triangles represent miRNAs, solid dots represent genes, and interactions are represented by straight lines.
The TYRP1 gene which regulates hair color production is highly conserved in function during evolution
Clustering analysis of TYR gene families in pigs, cattle, sheep, chickens, alpacas, and mice revealed that the TYR gene family belongs to a gene family shared by all six species (Fig. 3A). The hidden Markov model file (PF00264) of the TYR gene family was downloaded from the Pfam database. The sequence alignment of the swine protein sequence file identified a total of three TYR gene family members (TYR, TYRP1, and DCT) in pigs, while the Pfam database revealed the TYR gene family as an enzyme that promotes phenolic oxidation and is capable of catalyzing the production of melanin and other pigments by oxidation of tyrosine.
The distribution of TYR family genes on chromosomes was mapped using TBtools (Supplementary Fig S2A), and the results showed that the TYR family genes evolved without tandem replication events, resulting in the TYRP1, TYR, and DCT genes being located on chromosomes 1, 9 and 11 of the pig, respectively. ProtParam predicted basic physicochemical properties for members of the TYR gene family with amino acid sequence lengths ranging from 19444–87244aa and protein relative molecular masses ranging from 1635.39–7323.41 kDa. The isoelectric points (pI) ranged from 4.11–4.48, and all of the instability coefficients were greater than 40, indicating that all of the proteins in this family were unstable. The hydrophobicity values of all three proteins are greater than 0, indicating that all proteins in this family are hydrophobic (Supplementary Table S3).
To investigate the evolutionary relationship of TYR family genes within the pig species, multiple sequence alignment (Supplementary Fig S2B) was performed using TYR, TYRP1, and DCT protein sequence files, followed by the construction of an intraspecific phylogenetic tree (Fig. 3C), and the results of the intraspecific phylogenetic tree showed that TYR genes were the earliest and TYRP1 the latest in terms of evolutionary time relative to the other two genes.
As economic animals or model animals with hair color as an important phenotypic trait, cattle, sheep, alpacas, and mice were used as an outgroup to study the evolutionary relationship between them and TYR family genes in pigs, and the protein sequences of TYR family genes were used to construct an interspecific phylogenetic tree using the ornithischian chicken as an outgroup, and the results are shown in Fig. 3B. The TYR family genes in swine were located on three different-length evolutionary branches. Each evolutionary branch was for a different species of the same gene, indicating that TYRP1 and DCT were TYR paralogous homologs, and both TYRP1 and DCT evolved from TYR, and the affinities of the same gene varied among different species. The TYR family genes in swine were located on three different length evolutionary branches, each of which was for a different species of the same gene, indicating that TYRP1 and DCT were TYR paralogous genes, and both TYRP1 and DCT evolved from TYR, and the relatedness of the same gene varied among different species. Conservative motif analysis revealed that all members contained motifs 1–8, the distribution tended to be highly conserved, and gene family members shared almost identical sequences, maintaining a high degree of homogeneity in homogenous evolution (Fig. 3D). TYRP1 contains fewer motif 10 than TYR, and DCT contains fewer motif 9 than TYR, because TYR has accumulated mutations during the evolutionary process, causing some motif region sequences to change. In the gene structure map of the TYR family (Fig. 3E), the DCT gene contains 8 exonic regions, TYRP1 contains 6 exonic regions, and the TYR gene has the fewest exonic regions with only 5.
Gene replication events play an important role in the generation of novel functions and gene amplification. Therefore, in this study, the replication events of TYR genes in swine genomic species were analyzed (Fig. 3F). Intraspecific collinearity was found in the absence of tandem repeat events in swine TYR genes, likewise explaining the distribution of TYR family genes on different chromosomes. To speculate on the retention and loss of TYR, TYRP1, and DCT genes in species evolution. Collinearity analysis was performed on mammalian pigs, cattle, and ornithischian chickens, all of which resulted in three direct orthologous genes (Fig. 3G). The TYRP1 gene was transferred to chicken sex chromosome Z during evolution as a companion gene to control hair color formation, but remains on autosomal(chr12) in cattle. Both TYR and DCT genes are located on chromosome 1 in chickens, and correspondingly on chromosomes 29 and 8 in cattle, respectively.
The TYRP1 gene affects melanin production in Xiang pig melanocytes
In this experiment, the pEGFP-N3-TYRP1 eukaryotic expression vector (Supplementary Fig S3A) was constructed and successfully transfected into porcine melanocytes, and to detect the expression of TYR, TYRP1, and DCT genes at mRNA and protein levels. The qRT-PCR results showed that the relative mRNA expression levels of TYR, TYRP1, and DCT genes were significantly higher in the pEGFP-N3-TYRP1 transfected group compared with the pEGFP-N3 transfected group (P < 0.01) (Fig. 4A). The westen blotting results showed that the relative expression levels of TYR, TYRP1, and DCT proteins were significantly increased in the pEGFP-N3-TYRP1 transfected group compared with the pEGFP-N3 transfected group (P < 0.01) (Fig. 4B-C). It showed that overexpression of the TYRP1 gene could increase the expression of the TYR gene and DCT gene.
Five siRNAs of TYRP1 gene and control siRNA-NC were transfected with Xiang pig’s melanocytes separately for interference efficiency assay. The results showed that the mRNA expression of the TYRP1 gene was significantly decreased compared with the control (P < 0.01), with the highest interference efficiency of si4-TYRP1 (P < 0.01) (Supplementary Fig S3B). After successful transfection into melanocytes, the expression of downstream hair color-related genes was examined at the mRNA and protein levels. qRT-PCR results showed that the relative expression levels of TYR, TYRP1, and DCT genes were significantly decreased in the TYRP1-siRNA transfected group compared with the NC transfected group (P < 0.01) (Fig. 4D). The results of western blotting showed that the relative expression levels of TYR, TYRP1, and DCT proteins were significantly reduced in the TYRP1-siRNA transfected group compared with the NC transfected group (P < 0.01) (Fig. 4E-F). It indicated that reducing the expression of the TYRP1 gene could reduce the expression of the TYR gene and DCT gene.
Using the alkaline solubilization method to detect melanin content, the melanin content of the pEGFP-N3-TYRP1 transfected group was elevated 1.56-fold compared to the pEGFP-N3 transfected group (Fig. 4G), compared to the Negative Control transfected group, the melanin content of the TYRP1-siRNA transfected group was reduced by 0.763-fold (P < 0.01) compared with the Negative Control transfected group (Fig. 4H). The results of these data suggest that the TYRP1 gene can affect melanin production in melanocytes of Xiang pigs.
ssc-miR-221-3p targets the TYRP1 gene
The ssc-miR-221-3p was predicted to have a binding site to TYRP1 by the online software RNA22v2 (Fig. 5A), indicating that the TYRP1 gene may be one of the target genes of ssc-miR-221-3p. TYRP1 dual luciferase reporter vector (pmirGLO-TYRP1 CDS-wt) and mutant vector (pmirGLO-TYRP1 CDS-mut) were cotransfected into 293T cells with ssc-miR-221-3p mimic and Negative Control, respectively, and then subjected to dual luciferase reporter assay. The results showed that the luciferase activity of the pmirGLO-TYRP1 CDS-wt and ssc-miR-221-3p mimic cotransfected group was reduced by more than 30% compared with the pmirGLO-TYRP1 CDS-wt and Negative Control cotransfected group (Fig. 5B), which was highly significant (P < 0.01), while the difference with pmirGLO-TYRP1 CDS-mut and Negative Control cotransfected group did not differ significantly from the fluorescence activity of pmirGLO-TYRP1 CDS-mt and ssc-miR-221-3p mimic cotransfected group (Fig. 5C). It indicates that ssc-miR-221-3p can target the TYRP1 gene.
ssc-miR-221-3p targeted to inhibit the expression of TYRP1 gene and then affected melanogenesis in pig melanocytes.
After transfection of ssc-miR-221-3p mimic into melanocytes of Xiang pigs, qRT-PCR was applied to detect the expression of TYRP1, TYR, and DCT genes after transfection with ssc-miR-221-3p mimic, and the results showed that compared with the NC group, the expression levels of TYRP1, TYR and DCT mRNA expression levels were reduced by 87.21%, 38.41%, and 54.92%, respectively, with significant differences (Fig. 6B). It was discovered that ssc-miR-221-3p mimic could inhibit TYRP1 gene expression.
After ssc-miR-221-3p mimic was successfully transfected into Xiang pig melanocytes, the effect of ssc-miR-221-3p overexpression on melanogenesis-related genes was examined by Western blotting, and the results showed that compared with the NC group, the ssc-miR-221-3p group TYRP1, TYR, and TYRP2 protein expression levels were reduced by 42.22%, 27.69%, and 54.01%, respectively, with significant differences (Fig. 6A), indicating that ssc-miR-221-3p mimic could inhibit the expression of TYRP1 protein.
After ssc-miR-221-3p mimic was transfected with melanocytes from Xiang pigs, melanocytes from ssc-miR-221-3p mimic group and NC group were collected and counted separately. Melanin was extracted from melanocytes and the relative content of melanin was calculated. The results showed that the relative content of melanin in the ssc-miR-221-3p group decreased by 67.3% compared with that in the NC group, which was a significant difference (Fig. 6C). It showed that ssc-miR-221-3p mimic inhibited the expression of the TYRP1 gene and thus regulated melanin production in melanocytes of Xiang pigs by targeting action.