如皋黄鸡miR-1458与TBX6靶向关系验证  

Validation of Targeting Relationship Between miR-1458 and TBX6 in Rugao Yellow Chicken

作　　者：耿晴晴 程富富 胡菜 武嵘峰 吴宇慧 赵宗仪 赵梓多 张亚妮[1,2] GENG Qingqing;CHENG Fufu;HU Cai;WU Rongfeng;WU Yuhui;ZHAO Zongyi;ZHAO Ziduo;ZHANG Yani(Key Laboratory of Animal Genetic Breeding and Molecular Design,Jiangsu Province,College of Animal Science and Technology,Yangzhou University,Yangzhou,Jiangsu 225009;The Joint International Research Laboratory of Agriculture&Agri-Product Safety,Ministry of Education,Yangzhou University,Yangzhou,Jiangsu 225009)

机构地区：[1]扬州大学动物科学与技术学院,江苏省动物遗传繁育与分子设计重点实验室,江苏扬州225009 [2]扬州大学教育部农业与农产品安全国际合作联合实验室,江苏扬州225009

出　　处：《中国家禽》2023年第8期31-37,共7页China Poultry

基　　金：国家自然科学基金项目(32272857);扬州市国际合作项目(YZ2021175)。

摘　　要：研究旨在探究miR-1458与TBX6的靶向调控关系。试验基于前期建立视黄酸(Retinoic acid,RA)体外诱导胚胎干细胞(ESCs)向精原干细胞(SSCs)的分化模型,通过RNA-seq测序结果发现ESCs向SSCs分化过程中差异表达的miR-1458,采用miRNA定量检测miR-1458在ESCs/SSCs中的表达量,miR-1458靶基因富集通路分析筛选出富集通路,分析其与生殖细胞发育的相关性。基于前期联合分析RA诱导ESCs的mRNA-seq数据中|log2(fold change)|>8筛选出的与miR-1458互作的差异性mRNA,依据靶基因预测软件数据库在线预测该基因与miR-1458是否存在结合位点,利用qRT-PCR验证两者的关系,同时利用同源重组法将靶基因的3′UTR及点突变序列插入pMIR-REPORT^(TM)双荧光素酶报告载体中,共转染DF-1细胞,通过双荧光素酶报告载体确定miR-1458与TBX6的靶向调控关系。结果显示:ESCs向SSCs分化过程中差异表达miR-1458,miR-1458在生殖细胞分化过程中发挥重要作用;miR-1458与TBX63′UTR存在结合位点;miR-1458和TBX6分别在ESCs和SSCs中呈现此消彼长的表达趋势;miR-1458能够抑制TBX6的表达;miR-1458可靶向TBX63′UTR区调节TBX6的表达。研究表明miR-1458在鸡的ESCs和SSCs中呈现差异性高表达,并且miR-1458能够直接靶向调控TBX63′UTR区从而调节TBX6的表达,为后续研究miR-1458在SSCs形成过程中的机制探究奠定基础。This study is dedicated to investigate the targeting and regulatory relationship between miR-1458 and TBX6.Based on the previously established model of retinoic acid(RA)-induced differentiation of embryonic stem cells(ESCs)into sper⁃matogonial stem cells(SSCs)in vitro,RNA-seq sequencing results revealed that miR-1458 was differentially expressed during the differentiation of ESCs into SSCs.The expression of miR-1458 in ESCs/SSCs was quantitatively detected by quantitative detection of miRNA,enriched pathways were selected by miR-1458 target gene enrichment pathway analysis to analyze their correlation with germ cell development.Based on previous combined analysis of|log2(fold change)|>8 selected differential mRNAs interacting with miR-1458 in mRNA-seq data from RA-induced ESCs,the presence of binding sites between this gene and miR-1458 was predicted online based on the target gene prediction software database,and predicted whether there were binding sites between this gene and miR-1458 online according to the target gene prediction software database,and the relationship between the two was verified using qRT-PCR.At the same time,the 3′UTR and point mutation sequences of the target gene were inserted into the pMIR-REPORT^(TM) dual luciferase reporter vector using homologous recombination method and co-transfected into DF-1 cells,and the targeted regulation relationship between miR-1458 and TBX6 was determined by the dual luciferase reporter vector.The results showed that miR-1458 was differentially expressed in ESCs during differentiation into SSCs and miR-1458 played an im⁃portant role in germ cell differentiation,miR-1458 had a binding site with TBX63′UTR;miR-1458 and TBX6 showed a decreas⁃ing expression trend in ESCs and SSCs,respectively;miR-1458 could inhibit the expression of TBX6;miR-1458 could target the TBX63′UTR region to regulate the expression of TBX6.The results indicated that miR-1458 was differentially highly expressed in chicken ESCs and SSCs,and miR-1458 could directly target and regu

关 键 词：如皋黄鸡 miR-1458 TBX6 SSCs 双荧光素酶报告检测 

分 类 号：S831.2[农业科学—畜牧学]