为挖掘影响宁蒗高原鸡(Gallus gallus domestica)胫发育的信号通路和关键基因，本研究采集第6周龄(6W组)、12周龄(12W组)和18周龄(18W组)健康母鸡跖骨生长板组织进行转录组测序，筛选差异表达基因(differentially expressed gene，DEGs)并随机选取6个基因进行实时荧光定量PCR(real time quantitative PCR，RT-qPCR)验证。通过KEGG通路分类、KEGG和GO富集分析、蛋白质-蛋白质相互作用(protein-protein interaction，PPI)网络可视化分析，以及cytoHubba插件中的最大权团中心度(maximal clique centrality，MCC)、最大邻域分量密度(density of maximum neighborhood component，DMNC)和最大邻域分量(maximum neighborhood component，MNC)等3种算法，筛选出参与宁蒗高原鸡胫发育的候选信号通路和关键基因。结果表明，12W vs 6W组得到66个DEGs，上调DEGs 15个，下调DEGs 51个;18W vs 6W组得到1 814个DEGs，上调 DEGs 768 个，下调 DEGs 1 046 个;18W vs 12W 组得到 2 044 个 DEGs，上调 DEGs 1 049 个，下调 DEGs 995 个。6个基因的mRNA相对表达量变化趋势与转录组测序结果一致，表明测序结果可信。KEGG通路分类筛选出312个与生长发育相关的DEGs，KEGG富集分析进一步得到105条信号通路(P＜0。05)，GO富集分析表明信号转导、ATP结合、细胞外间隙和细胞外区域等条目被显著富集(P＜0。05)。综合KEGG和GO富集分析结果，得到15条信号通路和164个DEGs，PPI进一步分析筛选出可能参与宁蒗高原鸡胫发育的信号通路，根据表达量聚类和cytoHubba插件算法，得到调控胫发育的关键基因。本研究筛选到Wnt、TGF-β、PI3K-AKt、ECM-受体相互作用、细胞周期、黏着斑和刺猬共7条信号通路，以及IHH、COL9A1、COL6A2、FGF7、HGF和WNT4等6个基因为影响宁蒗高原鸡胫发育的候选信号通路和关键基因;并且IHH、COL9A1和WNT4基因在胫发育前期发挥作用，FGF7、HGF和COL6A2基因主要在胫发育后期发挥作用，IHH基因为影响宁蒗高原鸡胫发育的核心基因。该研究为探讨宁蒗高原鸡胫发育的分子机制提供理论支撑。
Screening Signal Pathways and Key Genes for Shank Length Trait of Ninglang Plateau Chickens Based on Transcriptome Sequencing
In order to explore the signaling pathways and key genes affecting the development of shank in Ninglang Plateau Chickens(Gallus gallus domestica),the metatarsal growth plate tissues of healthy hens at 6 weeks of age(6W group),12 weeks of age(12W group)and 18 weeks of age(18W group)were collected for transcriptome sequencing.The differentially expressed genes(DEGs)were screened and 6 genes were randomly selected for real time quantitative PCR(RT-qPCR)verification.Through KEGG pathway classifi-cation,KEGG and GO enrichment analysis,protein-protein interaction(PPI)network visualization analysis and 3 algorithms of maximal clique centrality(MCC),density of maximum neighborhood component(DMNC),and maximum neighborhood component(MNC)in cytoHubba plug-in,candidate signaling pathways and key genes involved in shank development of Ninglang Plateau chickens were screened out.The results showed that 66 DEGs were obtained in the 12W vs 6W group,15 DEGs were up-regulated and 51 DEGs were down-regulated.In the 18W vs 6W group,1 814 DEGs were obtained,768 DEGs were up-regulated and 1 046 DEGs were down-regula-ted.In the 18W vs 12W group,2 044 DEGs were obtained,1 049 DEGs were up-regulated and 995 DEGs were down-regulated.The change trend of mRNA relative expression of 6 genes was consistent with the results of transcriptome sequencing,indicating that the se-quencing results were credible.KEGG pathway classification screened 312 DEGs related to growth and development.KEGG enrichment analysis further obtained 105 signaling pathways(P＜0.05),and signal transduction,ATP binding,extracellular space,and extracellular region were significantly enriched in GO enrichment analysis(P＜0.05).Combining KEGG and GO enrichment analysis results,15 signa-ling pathways and 164 DEGs were obtained.PPI further analyzed and screened out signaling pathways that may be involved in the deve-lopment of shank in Ninglang Plateau chickens.According to the expression clustering and cytoHubba plug-in algorithm,the key genes regulating the development of shank were obtained.In this study,7 signal pathways,including Wnt,TGF-β,PI3K-AKt,ECM-receptor interaction,cell cycle,focal adhesion,and hedgehog,and 6 key genes,including IHH,COL9A1,COL6A2,FGF7,HGF,and WNT4,were selected as the candidate signaling pathways and key genes affecting the development of shank in Ninglang Plateau Chickens.Moreo-ver,IHH,COL9A1,and WNT4 play a role in the early stage of shank growth,while FGF7,HGF,and COL6A2 mainly play a role in the later stage of shank development.IHH gene is the core gene that affects the shank growth of Ninglang Plateau Chickens.This study can provide theoretical support for exploring the molecular mechanism of shank development in Ninglang Plateau Chickens.
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