巴什拜羊不同发育阶段骨骼肌中miR-486靶基因的研究

doi:10.3864/j.issn.0578-1752.2021.14.018

摘要/Abstract

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的表达量则呈相反的变化趋势,ARHGAP5和PDCD4在胎儿期和出生后巴什拜羊骨骼肌中的表达量未见显著变化。对其中4个候选靶基因的进一步试验验证,双荧光素酶报告载体试验结果表明,miR-486可以高效结合在其候选靶基因PTEN、Foxo1、IGF1R和PIK3R1的靶位点上,进而极显著抑制其后萤火虫荧光素酶的活性;巴什拜羊骨骼肌卫星细胞中的靶向调控试验结果亦表明miR-486可以显著下调上述4个候选靶基因的mRNA水平,抑制其生物学功能。所以可以确证在巴什拜羊骨骼肌中miR-486可以靶向调控PTEN、Foxo1、IGF1R和PIK3R1的表达。【结论】对巴什拜羊不同发育阶段骨骼肌中miR-486的靶基因进行了深入挖掘,全面解析了miR-486及其靶基因在巴什拜羊骨骼肌发育过程中参与的生物学过程和信号通路,所得候选靶基因数据可靠性高,可为阐明巴什拜羊优良肉质性状的分子调控机制奠定基础。

关键词: 巴什拜羊, miR-486, 靶基因, 骨骼肌, cDNA文库

Abstract:

【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 patterns were different. Among of them,PTEN, Foxo1, Dock3, PAX7, IGF1R, PIK3I1 and FBN1 were expressed at a relatively high level in the fetal period skeletal muscle of Bashbay sheep, but were significantly down regulated during postnatal period, and OLFM4 showed an opposite expression pattern compared with above 7 genes. The expression of ARHGAP5 and PDCD4 gene did not found significantly change in all 10 development periods of Bashbay sheep. The result of double luciferase reporter assay showed that miR-486 could bind the target sites of PTEN, Foxo1, IGF1R and PIK3I1 efficiently, and significantly suppressed the activity of firefly luciferase. In skeletal muscle satellite cell, mir-486 also could down-regulate mRNA of these four genes significantly, and finally suppressed their biology functions. So it was ultimately confirmed that miR-486 could regulate these 4 target genes indeed in skeletal muscle of Bashbay sheep. 【Conclusion】The investigation deeply revealed the target genes of miR-486 in skeletal muscle of Bashbay sheep during different development periods for the first time, and comprehensively analyzed the biology process and signaling pathway they participated. The further function assay proved that the data of target genes were reliable. These data would help to uncover the molecular regulation mechanisms related to excellent meat traits of Bashbay sheep.

Key words: Bashbay sheep, miR-486, target gene, skeletal muscle, cDNA library

张伟,王世银,高莉,杨力伟,邓双义,刘晓娜,石国庆,甘尚权. 巴什拜羊不同发育阶段骨骼肌中miR-486靶基因的研究[J]. 中国农业科学, 2021, 54(14): 3134-3148.

ZHANG Wei,WANG ShiYin,GAO Li,YANG LiWei,DENG ShuangYi,LIU XiaoNa,SHI GuoQing,GAN ShangQuan. Investigation of miR-486 Target Genes in Skeletal Muscle of Bashbay Sheep in Different Development Periods[J]. Scientia Agricultura Sinica, 2021, 54(14): 3134-3148.

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基因 Gene	登录号* Transcript ID	引物名称 Primers name	引物序列(5′-3′) Sequence of primers	片段大小 Size (bp)
PTEN	ENSOART00000015407.1	PTENqF	GTATTTGCAGTATAGAGCGTGC	165
PTEN	ENSOART00000015407.1	PTENqR	GGATTTGATGACTCCTCTACTG	165
Foxo1	ENSBTAT00000061127.3	Foxo1qF	CCACAGCAATGACGACTTCGAC	275
Foxo1	ENSBTAT00000061127.3	Foxo1qR	GACTGGGTGGACACAGTCAATG	275
Dock3	ENSOART00000008795.1	Dock3qF	AATCAGCCAAGCCTTCAGCTAG	246
Dock3	ENSOART00000008795.1	Dock3qR	TCTGCTCCCAGTCCATCATGTC	246
PAX7	ENSOART00000011524.1	PAX7qF	GTTCGATTAGCCGAGTGCTCAG	274
PAX7	ENSOART00000011524.1	PAX7qR	GTAGATGTCTGGGTAGTGGGTC	274
ARHGAP5	ENSOART00000006997.1	ARHG5qF	GTCTATCAGAACCATGTACAGC	189
ARHGAP5	ENSOART00000006997.1	ARHG5qR	CATACACCTCTTTGCTATCAGC	189
OLFM4	ENSOART00000007293.1	OLFM4qF	GACCATCTCCAAGAAGTTCGAG	159
OLFM4	ENSOART00000007293.1	OLFM4qR	CTTGATCAGCTCAAAGTCCAGC	159
IGF1R	ENSOART00000010895.1	IGF1RqF	GGAAGAGCTCGAGACTGAGTAC	159
IGF1R	ENSOART00000010895.1	IGF1RqR	GACAAAGTTAGAGGCGCTGCAG	159
PIK3R1	ENSOART00000006390.1	PIK3R1qF	CATGGGGATTACACTCTTACAC	147
PIK3R1	ENSOART00000006390.1	PIK3R1qR	CTAGAGATTCATTCCGGTAGTG	147
FBN1	ENSOART00000022901.1	FBN1qF	GGATTTCACGTCACACGAGATG	208
FBN1	ENSOART00000022901.1	FBN1qR	ACACAACGCCCATTCATGCAGA	208
PDCD4	ENSOART00000011353.1	PDCD4qF	ATTGCTAGAGCTGTTGGAGATG	250
PDCD4	ENSOART00000011353.1	PDCD4qR	TCAGCTTCAGAAATGTCTCCAG	250
β-actin	ENSOART00000003275.1	β-actin qF	TGTGCGTGACATCAAGGAGAAG	177
β-actin	ENSOART00000003275.1	β-actinqR	AGGAAGGAAGGCTGGAAGAG	177

引物 Primer	引物序列(5′-3′) Sequence of primers	片段大小Size (bp)	试验用途 Experiment
PTENcF	ccctcgagggCACCTTTCTTTAGCATGCTAC	262	载体构建 Vector construction
PTENcR	ccaagcttggGATAGCCTCCACATTTGTATG	262	载体构建 Vector construction
PTENmutF	GAATCTGTATTGGGGTACcttcATGAACCTTCCACAACAT	262	靶位点突变 Mutate bind site
PTENmutR	ATGTTGTGGAAGGTTCATgaagGTACCCCAATACAGATTC	262	靶位点突变 Mutate bind site
Foxo1cF	ccctcgagggGAGAAGCAGTCCAAAGATGTC	221	载体构建 Vector construction
Foxo1cR	ccaagcttggATGGTGTAGTGAGTTTGGCAC	221	载体构建 Vector construction
Foxo1mutF	CGAAGACGCTTCCTGTACcttcTGTTTGCCCAGTGTTTGC	221	靶位点突变 Mutate bind site
Foxo1mutR	GCAAACACTGGGCAAACAgaagGTACAGGAAGCGTCTTCG	221	靶位点突变 Mutate bind site
IGF1RcF	ccctcgagggGAGAATCCCAATGGATTGATC	237	载体构建 Vector construction
IGF1RcR	ccaagcttggGGATGAAGTTCTCATATGTCG	237	载体构建 Vector construction
IGF1RmutF	GTGTCCAGACAGGAGTACcttcAGTATGGAGGAGCCAAGC	237	靶位点突变 Mutate bind site
IGF1RmutR	GCTTGGCTCCTCCATACTgaagGTACTCCTGTCTGGACAC	237	靶位点突变 Mutate bind site
PIK3R1cF	ccctcgagggCTGAAGGCTAAATTCACAGTG	267	载体构建 Vector construction
PIK3R1cR	ccaagcttggTAACAGCCAAGTACTCTGTAC	267	载体构建 Vector construction
PIK3R1mutF	GCTTTTAAAGAAATGTACcttcTGCCAGTTTGTCAAGTCG	267	靶位点突变 Mutate bind site
PIK3R1mutR	CGACTTGACAAACTGGCAgaagGTACATTTCTTTAAAAGC	267	靶位点突变 Mutate bind site

名称 Name	序列(5′-3′) Sequence
miR-486 mimics	UCCUGUACUGAGCUGCCCCGAG
miR-486 mimics negative control	CAGUACUAGCGUGUAGUACCAA
miR-486 inhibitor	CUCGGGGCAGCUCAGUACAGGA
miR-486 inhibitor negative control	GCAUGGCUUGAGUCCGUAAGAU