摘要
【目的】探索鸡miR-203a在黑色素沉积过程中潜在的作用机制。【方法】通过miRbase数据库获取miR-203a的成熟体序列,利用MEGA11软件构建miR-203a的系统进化树,采用TargetScan和miRDB在线工具预测鸡miR-203a的靶基因,并对其进行GO和KEGG富集分析;利用实时荧光定量PCR(quantitative real-time polymerase chain reaction,RT-qPCR)检测miR-203a在淅川乌骨鸡和固始鸡背肤,以及在淅川乌骨鸡不同组织或器官中的表达水平;在黑色素细胞中对miR-203a进行过表达和干扰后,利用RT-qPCR检测miR-203a的靶基因丝裂原活化蛋白激酶10基因(mitogen-activated protein kinase 10,MAPK10)的表达水平。【结果】miR-203a在不同物种间高度保守,经系统进化树序列比对分析,鸡miR-203a与斑马鱼的亲缘关系较近,与大鼠和人亲缘关系较远,预测得到鸡miR-203a有150个靶基因。GO功能富集分析表明,miR-203a靶基因主要富集在基因表达调控、生长因子应答和大分子代谢过程调控等过程;KEGG通路富集显示,miR-203a靶基因显著富集于MAPK信号通路、焦点黏附、昼夜节律、Wnt信号通路等。miR-203a在固始鸡背肤中的表达水平显著高于淅川乌骨鸡;通过淅川乌骨鸡不同组织或器官表达谱分析,miR-203a在背肤中的表达水平最高,肾脏和心脏次之,均显著高于肝脏和脾脏。在黑色素细胞中过表达miR-203a,靶基因MAPK10表达水平显著下降,干扰miR-203a后,MAPK10表达水平显著上升。【结论】miR-203a在物种进化过程中高度保守,在黑色素细胞中起负调控作用,通过负调控MAPK10基因进而调节MAPK及Wnt等多条信号通路。
【Objective】The objective of this study is to explore the potential mechanism of miR-203a in the process of melanin deposition in chickens.【Method】The mature sequence of miR-203a was obtained from miRbase database,and MEGA11 software was used to construct the phylogenetic tree of miR-203a.The target genes of miR-203a were predicted using the online tools of TargetScan and miRDB,and GO and KEGG enrichment analysis were performed.The expression levels of miR-203a in the dorsal skin of Xichuan Black-bone chickens and Gushi chickens,as well as in various tissues or organs of Xichuan Black-bone chickens,were detected using quantitative real-time polymerase chain reaction(RT-qPCR).After overexpression and interference of miR-203a in melanocytes,the expression levels of its target gene,mitogen-activated protein kinase 10(MAPK10),were measured by RTqPCR.【Result】miR-203a is highly conserved among different species,and phylogenetic tree sequence comparison analysis shows that chickens are closely related to zebrafish,but they are more distantly related to rats and humans.A total of 150 target genes of miR-203a were predicted by two databases.GO functional enrichment analysis showed that target genes were mainly enriched in gene expression regulation,growth factor response and macromolecular metabolism regulation.KEGG pathway enrichment analysis revealed that miR-203a target genes were significantly enriched in the MAPK signaling pathway,focal adhesion,circadian rhythm,Wnt signaling pathway,and others.The expression level of miR-203a in the back skin of Gushi chickens was significantly higher than that in Xichuan black bone chickens.In Xichuan black bone chicken,the relative expression level of miR-203a was the highest in back skin,followed by kidney and heart,which were significantly higher than liver and spleen.Overexpression of miR-203a in melanocytes significantly increased the expression level of the target gene MAPK10(p<0.05),and interference with miR-203a significantly increased the expression level of MAPK10(p<0.05).【Conclusion】miR-203a is highly conserved across species evolution and plays a negative regulatory role in melanocytes.It modulates multiple signaling pathways,including MAPK and Wnt,by negatively targeting the MAPK10 gene.
作者
耿晓文
徐硕辉
张志远
侯英东
何富民
李闯
李文婷
李东华
GENG Xiaowen;XU Shuohui;ZHANG Zhiyuan;HOU Yingdong;HE Fumin;LI Chuang;LI Wenting;LI Donghua(College of Animal Science and Technology,Henan Agricultural University,Zhengzhou 450046,China;Henan Key Laboratory of Chicken Germplasm Resources Innovation and Utilization,Zhengzhou 450046,China)
出处
《河南农业大学学报》
北大核心
2025年第3期434-443,共10页
Journal of Henan Agricultural University
基金
国家自然科学基金青年项目(32102540)
河南农业大学青年科技创新基金项目(KJCX2021A09)。
关键词
淅川乌骨鸡
miR-203a
进化分析
靶基因
表达分析
Xichuan black bone chicken
miR-203a
evolutionary analysis
target gene function prediction
expression analysis
