基于转录组测序的巴马香猪和杜洛克猪骨骼肌差异lncRNA分析

Differential lncRNA analysis of skeletal muscles in Bama Xiang pig and Duroc pig based on transcriptome sequencing

【目的】通过对巴马香猪和杜洛克猪背最长肌组织进行转录组测序鉴定差异表达lncRNA,筛选与骨骼肌发育形成相关的候选基因,为明确lncRNA对骨骼肌生长发育的调控机制提供理论基础。【方法】采集12月龄的巴马香猪和杜洛克猪背最长肌组织进行转录组测序,以错误发现率(FDR)<0.05且|log2Fold Change|>1为标准筛选差异表达基因(DEGs)和差异表达lncRNA,并进行实时荧光定量PCR验证,预测差异表达lncRNA靶基因并进行GO功能注释和KEGG信号通路富集分析,选取表达差异最大的lncRNA构建其与靶基因互作网络。【结果】在巴马香猪和杜洛克猪背肌间共鉴定出6316个DEGs和675个差异表达lncRNA;GO功能注释分析结果表明,差异表达lncRNA靶基因主要涉及代谢过程、肌肉组织发育及骨骼肌细胞增殖和分化等生物过程;KEGG信号通路富集分析结果表明,差异表达lncRNA靶基因在Hippo和Wnt信号通路显著富集(P<0.05),说明差异表达lncRNA与骨骼肌细胞的增殖、自噬和分化相关;对表达差异最大的lncRNA-MSTRG.16703进行实时荧光定量PCR验证,发现其在巴马香猪中的相对表达量极显著高于杜洛克猪(P<0.01),与转录组测序结果一致。lncRNA-MSTRG.16703与靶基因互作网络分析结果表明,上调靶基因包括QKI、MBNL1和YBX2,QKI和MBNL1在均与可变剪接相关。【结论】在巴马香猪和杜洛克猪间发现的差异表达lncRNA是调控骨骼肌发育的候选基因,其靶基因主要富集在Hippo和Wnt等骨骼肌发育相关信号通路。lncRNA-MSTRG.16703的表达差异最大且在巴马香猪中上调,其靶基因在骨骼肌细胞增殖分化和肌纤维形成中有重要调控作用。

【Objective】The aim of this study was to provide a theoretical basis for clarifying the regulatory mechanism of lncRNA on skeletal muscle growth and development,by conducting transcriptome sequencing on the longest dorsal muscle tissues of Bama Xiang pigs and Duroc pigs to identify differential expression of lncRNA and screening candidate genes related to skeletal muscle development.【Method】Transcriptome sequencing was performed on the longest dorsal muscle tissues of 12 months-old Bama Xiang pigs and Duroc pigs.Using false discovery rate(FDR)<0.05 and|log2 Fold Change|>1 as the standard,differentially expressed genes(DEGs)and differentially expressed lncRNA were screened,and real-time fluorescence quantitative PCR validation was performed,in order to predict differentially expressed lncRNA target genes and to perform GO functional annotation and KEGG signaling pathway enrichment analysis.The lncRNA with the greatest differential expression was selected to construct its interaction network with target genes.【Result】The results showed that a total of 6316 DEGs and 675 differentially expressed lncRNA were identified in the dorsal muscles of Bama Xiang pigs and Duroc pigs.GO functional annotation analysis showed that differentially expressed lncRNA target genes mainly involved biological processes such as metabolism process,muscle tissue development,and skeletal muscle cell proliferation and differentiation.KEGG signaling pathway enrichment analysis showed that differentially expressed lncRNA target genes were significantly enriched in the Hippo and Wnt signaling pathways(P<0.05),indicating that differentially expressed lncRNA was associated with proliferation,autophagy,and differentiation of skeletal muscle cells.Real time fluorescence quantitative PCR validation was performed on lncRNA-MSTRG.16703,which had the largest expression difference.It was found that its relative expression level in Bama Xiang pigs was extremely significantly higher than that in Duroc pigs(P<0.01),which was consistent with the result of transcriptom sequencing.Analysis of the interaction network between lncRNA-MSTRG.16703 and target genes showed that the up-regulated target genes including QKI,MBNL1,and YBX2.QKI and MBNL1 were related to alternative splicing.【Conclusion】The differentially expressed lncRNA found between Bama Xiang pig and Duroc pig is a candidate gene for regulating skeletal muscle development,its target genes mainly enrich on skeletal muscle development related signaling pathways such as Hippo and Wnt.The expression difference of lncRNA-MSTRG.16703 is the largest and up-regulates in Bama Xiang pigs.Its target gene plays an important regulatory role in the proliferation and differentiation of skeletal muscle cells and muscle fiber formation.