乌头驴与三粉驴骨骼肌中miRNAs的表达鉴定与分析

Identification and Analysis of miRNAs Expression in Skeletal Muscle of Wutou Donkey and Sanfen Donkey

试验旨在分析microRNAs(miRNAs)在德州驴骨骼肌中的表达情况,探讨小RNA(small RNA)在驴骨骼肌发育中的调控机制。采集德州驴2个品系(乌头驴和三粉驴)的背最长肌组织,构建乌头驴背最长肌(WB)和三粉驴背最长肌(SB)的small RNA测序文库并进行测序,运用生物信息学技术对WB和SB之间差异表达的miRNAs进行分析,预测和解析可能影响驴产肉性状的miRNAs,从中挑选了6个表达量较高的miRNAs,利用实时荧光定量PCR技术来验证测序结果的准确性。结果表明,在WB和SB文库中分别鉴定出保守miRNAs有982和1149个,新miRNAs有106和143个。在WB与SB比较组中基于条件|log2(fold_change)|≥2、P≤0.05获得17个差异表达的miRNAs,其中表达量上调的有5个,下调的有12个。通过GO注释,发现差异表达的miRNAs显著富集于调控细胞迁移分化、细胞分裂和骨骼肌细胞收缩等生物学过程(P<0.05),并且挖掘到与驴骨骼肌发育相关的bta-miR-378d_L+1R-1_1ss22CA、bta-miR-378d_R-2,其靶向NPY1R、GPR152、BEST1、KCNH8、SPAG9等多个基因。KEGG富集结果表明,miRNAs富集于Wnt、MAPK、泛素蛋白水解系统等与骨骼肌发育相关的信号通路中,其中在Wnt信号通路中共涉及到17个差异表达miRNAs的207个靶基因,尤其以PC-5p-84483_31、hsa-miR-186-3p_R-3和cfa-miR-329b_1ss19CT的靶基因最多。实时荧光定量PCR结果与miRNAs测序结果一致,并发现除eca-miR-181b外,其他5个miRNAs在SB中的表达水平均高于WB。本研究首次对驴骨骼肌miRNAs进行转录组学分析,揭示了乌头驴和三粉驴miRNAs的表达差异,研究结果为阐明德州驴骨骼肌生长发育的分子调控机制提供参考。

To investigate the expression of microRNAs(miRNAs)in skeletal muscles of Dezhou donkeys and further improve the regulatory mechanism of small RNA in the development of skeletal muscle of donkey.In this study,the longissimus dorsi muscle of two Dezhou donkey strains(Wutou donkey and Sanfen donkey)was selected as the research objects,by constructing small RNA sequencing library of Wutou donkey longissimus dorsi muscle(WB)and Sanfen donkey longissimus dorsi muscle(SB),bioinformatics techniques were used to carry out differential expression analysis of miRNAs between WB and SB,and predict and analyze miRNAs that might affect the meat production traits of donkey.In addition,six miRNAs with high expression were selected,and Real-time quantitative PCR was used to verify the accuracy of sequencing results.The results showed that 982 and 1149 conserved miRNAs and 106 and 143 new miRNAs were identified in WB and SB libraries.In WB and SB comparison group(WB vs SB)based on conditions|log2(fold_change)|≥2,P≤0.05 get 17 differentially expressed miRNAs,of which 5 were up-regulated and 12 were down-regulated.Through GO annotation,it found that differentially expressed miRNAs were significantly enriched in biological processes such as regulating cell migration and differentiation,cell division and skeletal muscle cell contraction(P<0.05).bta-miR-378d_L+1R-1_1ss22CA and bta-miR-378d_R-2 which related to skeletal muscle development in donkey was found,they target multiple genes such as NPY1R,GPR152,BEST1,KCNH8 and SPAG9.KEGG enrichment results showed that miRNAs were enriched in Wnt,MAPK,ubiquitin proteolysis system and other signaling pathways related to skeletal muscle development.Among them,a total of 207 target genes 17 differentially expressed miRNAs were involved in the Wnt signaling pathway,especially PC-5p-84483_31,hsa-miR-186-3p_R-3 and cfa-miR-329b_1ss19CT had the largest number of target genes.The results of Real-time quantitative PCR were consistent with the results of miRNAs sequencing,and it found that the expression levels of the other five miRNAs were all higher in the SB than in the WB except for eca-miR-181b.In this study,miRNAs of skeletal muscle of two strains of Dezhou donkey were analyzed for the first time,and the differences of RNA-seq between Wutou donkey and Sanfen donkey were revealed,which provided reference for elucidating the molecular regulation mechanism of skeletal muscle growth and development of Dezhou donkey.