基于转录组测序的剑白香猪表皮颜色相关miRNA筛选与鉴定

Screening and identification of miRNAs related to epidermal color in Jianbai Xiang pigs based on transcriptome sequencing

【目的】对剑白香猪黑色和白色表皮组织进行转录组测序,筛选出对黑色素生成起调节作用的miRNA,为进一步研究miRNA对剑白香猪表皮颜色形成的调控机制提供理论依据。【方法】以剑白香猪黑色和白色表皮组织为试验材料进行转录组测序,以|log2FoldChange|≥1且错误发现率(FDR)≤0.05为标准,采用DESeq2筛选差异表达miRNA,使用miRanda和RNAhybrid预测差异表达miRNA靶基因并进行GO功能注释和KEGG信号通路富集分析,构建差异表达miRNA、靶基因和KEGG信号通路互作网络,利用实时荧光定量PCR对转录组测序结果进行验证。【结果】从剑白香猪黑色和白色表皮中鉴定出280个已知miRNA和618个新miRNA,筛选出17个差异表达miRNA,白色表皮较黑色表皮有10个miRNA上调表达,7个miRNA下调表达。共预测获得1327个靶基因,GO功能注释分析结果显示,在生物学过程中,差异表达miRNA靶基因主要涉及上皮发育、细胞增殖和分解代谢过程等条目;在细胞组分和分子功能中,主要涉及细胞质、细胞质囊泡、ATP结合和催化活性等条目。KEGG信号通路富集结果显示,靶基因在磷脂酰肌醇信号系统、黑色素生成和细胞色素P450等信号通路富集,其中ssc-miR-615靶基因包括CALM3、CREBBP、FZD5和DVL2,其富集通路均与表皮色素沉着有关,ssc-miR-221-3p靶基因TYRP1和ssc-miR-224靶基因RAB23富集的通路与黑色素生成有关。选取7个差异表达miRNA与靶基因TYRP1和RAB23进行实时荧光定量PCR验证,结果表明差异表达miRNA的表达变化与转录组测序分析的变化趋势一致,TYRP1和RAB23在剑白香猪黑色表皮中的表达量极显著高于白色表皮(P<0.01)。【结论】ssc-miR-615、ssc-miR-221-3p和ssc-miR-224是影响剑白香猪表皮颜色的候选基因,可能对剑白香猪表皮的黑色素形成有重要影响。

【Objective】The purpose of the study was to sequence the transcriptome of black and white epidermal tissues of Jianbai Xiang pig,to screen out miRNA that regulated melanin production,and to provide a theoretical basis for further study on the regulatory mechanism of miRNA on epidermal color formation of Jianbai Xiang pig.【Method】The black and white epidermal tissues of Jianbai Xiang pigs were used as experimental materials for transcriptome sequencing.DESeq2 was used to screen differentially expressed miRNAs with|log2 Fold Change|≥1 and false discovery rate(FDR)≤0.05 as the standard.The target genes of differentially expressed miRNAs were predicted by miRanda and RNAhybrid,and the enrichment analysis of KEGG signaling pathway was performed by GO functional annotation.The interaction network of differentially expressed miRNAs,target genes and KEGG signaling pathway was constructed,and the results of transcriptome sequencing were verified by real-time fluorescence quantitative PCR.【Result】A total of 280 known miRNAs and 618 new miRNAs were identified from the black and white epidermis of Jianbai Xiang pig,and 17 differentially expressed miRNAs were screened.Ten miRNAs were up-regulated and seven miRNAs were down-regulated in the white epidermis compared with the black epidermis.A total of 1327 target genes were predicted and obtained.GO functional annotation showed that in biological processes,the differentially expressed miRNA target genes were mainly involved in epithelial development,cell proliferation and catabolic processes.In cell components and molecular functions,they were mainly involved in cytoplasm,cytoplasmic vesicles,ATP binding and catalytic activity.The enrichment results of KEGG signaling pathway showed that the target genes were enriched in phosphatidylinositol signaling system,melanin production and cytochrome P450 signaling pathways.Among them,ssc-miR-615 target genes included CALM3,CREBBP,FZD5 and DVL2,and their enrichment pathways were all related to epidermal pigmentation.The enrichment pathways of ssc-miR-221-3p target gene TYRP1 and ssc-miR-224 target gene RAB23 were related to melanin production.Seven differentially expressed miRNAs and target genes TYRP1 and RAB23 were selected for real-time fluorescence quantitative PCR verification.The results showed that the expression changes of differentially expressed miRNAs were consistent with the changing trend of transcriptome sequencing analysis.The expression of TYRP1 and RAB23 in the black epidermis of Jianbai Xiang pigs was extremely significantly higher than that in the white epidermis(P<0.01).【Conclusion】The ssc-miR-615,ssc-miR-221-3p and ssc-miR-224 are candidate genes affecting the epidermal color of Jianbai Xiang pig,which may have an important effect on the formation of melanin in the epidermis of Jianbai Xiang pig.