Table_7_Comparison of differentially expressed genes in longissimus dorsi muscle of Diannan small ears, Wujin and landrace pigs using RNA-seq.XLSX

IntroductionPig growth is an important economic trait that involves the co-regulation of multiple genes and related signaling pathways. High-throughput sequencing has become a powerful technology for establishing the transcriptome profiles and can be used to screen genome-wide differentially expressed genes (DEGs). In order to elucidate the molecular mechanism underlying muscle growth, this study adopted RNA sequencing (RNA-seq) to identify and compare DEGs at the genetic level in the longissimus dorsi muscle (LDM) between two indigenous Chinese pig breeds (Diannan small ears [DSE] pig and Wujin pig [WJ]) and one introduced pig breed (Landrace pig [LP]).
MethodsAnimals under study were from two Chinese indigenous pig breeds (DSE pig, n = 3; WJ pig, n = 3) and one introduced pig breed (LP, n = 3) were used for RNA sequencing (RNA-seq) to identify and compare the expression levels of DEGs in the LDM. Then, functional annotation, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein–Protein Interaction (PPI) network analysis were performed on these DEGs. Then, functional annotation, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) network analysis were performed on these DEGs.
ResultsThe results revealed that for the DSE, WJ, and LP libraries, more than 66, 65, and 71 million clean reads were generated by transcriptome sequencing, respectively. A total of 11,213 genes were identified in the LDM tissue of these pig breeds, of which 7,127 were co-expressed in the muscle tissue of the three samples. In total, 441 and 339 DEGs were identified between DSE vs. WJ and LP vs. DSE in the study, with 254, 193 up-regulated genes and 187, 193 down-regulated genes in DSE compared to WJ and LP. GO analysis and KEGG signaling pathway analysis showed that DEGs are significantly related to contractile fiber, sarcolemma, and dystrophin-associated glycoprotein complex, myofibril, sarcolemma, and myosin II complex, Glycolysis/Gluconeogenesis, Propanoate metabolism, and Pyruvate metabolism, etc. In combination with functional annotation of DEGs, key genes such as ENO3 and JUN were identified by PPI network analysis.
DiscussionIn conclusion, the present study revealed key genes including DES, FLNC, PSMD1, PSMD6, PSME4, PSMB4, RPL11, RPL13A, ROS23, RPS29, MYH1, MYL9, MYL12B, TPM1, TPM4, ENO3, PGK1, PKM2, GPI, and the unannotated new gene ENSSSCG00000020769 and related signaling pathways that influence the difference in muscle growth and could provide a theoretical basis for improving pig muscle growth traits in the future.

引言
猪生长是受多基因及相关信号通路共同调控的重要经济性状。高通量测序已成为构建转录组图谱、筛选全基因组差异表达基因（differentially expressed genes, DEGs）的有力技术手段。为阐明肌肉生长的分子机制，本研究采用RNA测序（RNA-seq），在遗传层面鉴定并比较3个猪群背最长肌（longissimus dorsi muscle, LDM）中的差异表达基因：2个中国地方猪品种——滇南小耳猪（Diannan small ears, DSE）、乌金猪（Wujin pig, WJ），以及1个引进猪品种长白猪（Landrace pig, LP）。
方法
本研究选取2个中国地方猪品种（滇南小耳猪，n=3；乌金猪，n=3）与1个引进猪品种长白猪（n=3）的个体作为实验材料，对其背最长肌组织进行RNA测序，以鉴定并比较其中的差异表达基因表达水平。随后对这些差异表达基因开展功能注释、基因本体（Gene Ontology, GO）富集分析、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析，以及蛋白质-蛋白质相互作用（Protein–Protein Interaction, PPI）网络分析。随后对这些差异表达基因开展功能注释、基因本体（Gene Ontology, GO）富集分析、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析，以及蛋白质-蛋白质相互作用（Protein–Protein Interaction, PPI）网络分析。
结果
转录组测序结果显示，滇南小耳猪、乌金猪与长白猪的测序文库分别产生了超过6600万、6500万与7100万条清洁读段（clean reads）。在上述猪品种的背最长肌组织中共鉴定到11213个基因，其中7127个基因在3个群体的肌肉组织中均有共表达。本研究中，滇南小耳猪vs乌金猪、长白猪vs滇南小耳猪的比较组分别鉴定到441个和339个差异表达基因；与乌金猪、长白猪相比，滇南小耳猪分别有254个、193个上调基因，以及187个、193个下调基因。基因本体富集分析与京都基因与基因组百科全书信号通路分析显示，差异表达基因显著富集于收缩纤维、肌膜、抗肌萎缩蛋白相关糖蛋白复合物、肌原纤维、肌球蛋白II复合物，以及糖酵解/糖异生、丙酸代谢、丙酮酸代谢等通路。结合差异表达基因的功能注释，通过蛋白质相互作用网络分析鉴定到ENO3与JUN等关键基因。
讨论
综上，本研究揭示了影响肌肉生长差异的关键基因，包括DES、FLNC、PSMD1、PSMD6、PSME4、PSMB4、RPL11、RPL13A、ROS23、RPS29、MYH1、MYL9、MYL12B、TPM1、TPM4、ENO3、PGK1、PKM2、GPI，以及未注释新基因ENSSSCG00000020769和相关信号通路，可为未来改良猪肌肉生长性状提供理论依据。