巴什拜羊不同发育阶段骨骼肌中miR-486靶基因的研究  

Investigation of miR-486 Target Genes in Skeletal Muscle of Bashbay Sheep in Different Development Periods

作　　者：张伟[1,2] 王世银[1] 高莉[1] 杨力伟[1] 邓双义[1] 刘晓娜[1] 石国庆[2] 甘尚权[2] ZHANG Wei;WANG ShiYin;GAO Li;YANG LiWei;DENG ShuangYi;LIU XiaoNa;SHI GuoQing;GAN ShangQuan(Xinjiang Agricultural Professional Technological College,Changji 831100,Xinjiang;State Key Laboratory of Sheep Genetic Improvement and Healthy Production/Institute of Animal Husbandry and Veterinary,Xinjiang Academy of Agricultural and Reclamation Sciences,Shihezi 832000,Xinjiang)

机构地区：[1]新疆农业职业技术学院,新疆昌吉831100 [2]新疆农垦科学院畜牧兽医研究所/省部共建绵羊遗传改良与健康养殖重点实验室,新疆石河子832000

出　　处：《中国农业科学》2021年第14期3134-3148,共15页Scientia Agricultura Sinica

基　　金：新疆维吾尔自治区自然科学基金面上项目(2017D01A51)。

摘　　要：【目的】为了深入挖掘巴什拜羊不同发育阶段骨骼肌中miR-486的靶基因,为最终阐明巴什拜羊优良肉质性状形成的分子调控机制奠定基础,同时为巴什拜羊的持续选育提供理论依据。【方法】分别采集了巴什拜羊胎儿期40,50,60,80,100和120 d,以及出生当天,出生后30,60和90 d共计10个不同发育阶段的骨骼肌组织,分别利用胎儿期6个阶段的混合mRNA和出生后4个阶段的混合mRNA构建了胎儿期和出生后骨骼肌中miR-486调控靶基因的cDNA文库,并利用高通量测序技术对cDNA文库中的靶基因信息进行了深入挖掘。在对所得候选靶基因功能分析的基础上,选择10个候选靶基因,使用qRT-PCR检测其在巴什拜羊上述10个不同发育阶段骨骼肌中表达量的变化,结合课题组前期获得的miR-486在巴什拜羊不同发育阶段骨骼肌中的表达规律,对其靶向调控关系及生物学功能进行初步验证和分析。进而选择其中的4个候选靶基因使用荧光素酶报告载体试验和巴什拜羊骨骼肌卫星细胞中的靶向调控试验最终确认其靶向调控关系。【结果】通过对两个cDNA文库高通量测序数据的分析,胎儿期和出生后分别获得了123个和118个miR-486的靶基因,因其靶向调控关系未经实验验证,故暂称为候选靶基因。其中有96个为两个阶段共表达的候选靶基因,其余27个和22个分别为胎儿期和出生后骨骼肌中特异表达的候选靶基因。GO和KEGG分析结果表明所得候选靶基因显著富集于与肌肉分化和肌纤维发育相关的代谢和信号转导通路,诸如PI3k-Akt、MAPK、Wnt、Adherens junction和Regulation of actin cytoskeleton等。qRT-PCR检测结果表明所选10个候选靶基因在不同发育阶段巴什拜羊骨骼肌中均有表达,但其表达变化规律有所不同。PTEN、Foxo1、Dock3、PAX7、IGF1R、PIK3I1和FBN1在胎儿期巴什拜羊骨骼肌中呈高表达,出生后其表达量显著下调,而OLFM4的表达量则�【Objective】The aim of this study was to deeply reveal the target genes of miR-486 in skeletal muscle of Bashbay sheep during different development periods,and to get basic data for finally uncover the molecular regulation mechanism of excellent meat traits of this sheep breed,then finally to support to further breeding.【Method】The skeletal muscle of Bashbay sheep were collected during 40 d,50 d,60 d,80 d,100 d and 120 d of fetal period,and 1,2,and 3 months old of new born sheep.The total RNA was extracted,and two cDNA libraries of miR-486 target genes were constructed using mRNA of fetal and postnatal period,respectively,then the library was sequenced applied high-throughput sequencing technology.Based on the function analysis,10 candidate target genes were selected,and their express patterns in Bashbay sheep skeletal muscle of 10 development periods mentioned above were detected by qRT-PCR.By analyzing the expression pattern of these 10 candidate target genes and miR-486 in skeletal muscle of Bashbay sheep,their target regulation relationship and functions were primarily verified.Finally,4 candidate target genes were chosen to confirm their regulation relationship using the double luciferase reporter assay and target regulation assay in satellite cell of skeletal muscle of Bashbay sheep.【Result】Total 123 and 118 target genes of fetal and postnatal period were obtained,respectively.Due to these target genes were not confirmed by further function assay,so they were called candidate target genes temporarily.Among of them,96 genes were expressed in skeletal muscle of fetal and postnatal period,27 and 22 genes were specially expressed in these two periods respectively.The result of GO and KEGG analysis showed that these candidate target genes regulated mass pathways related to muscle cell differentiation and development,just like PI3k-Akt,MAPK,Wnt,Adherens junction and Regulation of actin cytoskeleton,etc.All 10 candidate target genes were expressed in skeletal muscle of Bashbay sheep,but their expression

关 键 词：巴什拜羊 miR-486 靶基因 骨骼肌 CDNA文库 

分 类 号：S826[农业科学—畜牧学]