二花脸猪linc-NORFA核心启动子鉴定与转录调控分析

doi:10.3864/j.issn.0578-1752.2021.15.016

摘要/Abstract

摘要：

【目的】前期研究证实linc-NORFA作为母猪繁殖性状候选基因参与调控卵泡闭锁与卵泡颗粒细胞凋亡过程,进一步鉴定二花脸猪linc-NORFA核心启动子并分析其转录调控机制,为解析linc-NORFA介导猪卵泡闭锁的分子机制奠定理论基础并提供新的研究思路。【方法】采集二花脸猪耳组织样品并提取基因组DNA,利用PCR扩增和测序技术获得二花脸猪linc-NORFA 5′调控区序列;通过构建缺失表达荧光报告载体并利用荧光素酶活性试验鉴定二花脸猪linc-NORFA核心启动子;利用生物信息学分析二花脸猪linc-NORFA核心启动子区序列特征与潜在的转录因子结合位点;构建猪FOXO1真核生物表达载体并进一步采用Western blot、qRT-PCR以及荧光素酶活性试验分析转录因子FOXO1过表达对二花脸猪linc-NORFA转录的影响;利用染色质免疫沉淀（ChIP）技术鉴定转录因子FOXO1与二花脸猪linc-NORFA核心启动子区的结合能力。【结果】通过克隆测序与序列拼接共获得二花脸猪linc-NORFA 5′调控区序列1 734 bp,其中包含两个潜在的CpG岛;利用荧光素酶活性试验证实linc-NORFA核心启动子位于-988 — -684 bp（转录起始位点作为+1）,生物信息学分析表明二花脸猪linc-NORFA核心启动子上包含多个转录因子的结合元件,例如ESR2、FOXO1、E2F1、BRCA1以及NFIC等;另外,ChIP试验还证实在猪卵巢颗粒细胞中FOXO1作为转录因子直接靶向结合在linc-NORFA的核心启动子区;进一步通过试验证实FOXO1过表达可显著下调linc-NORFA核心启动子区活性（P<0.01）,同时显著抑制体外培养的猪卵巢颗粒细胞中linc-NORFA的表达（P<0.01）。【结论】鉴定了二花脸猪linc-NORFA核心启动子区,同时证实FOXO1作为转录因子能够与linc-NORFA核心启动子区特异性结合,进而抑制后者的转录活性与表达。研究结果对探究linc-NORFA在猪卵泡闭锁过程中显著下调的分子机制具有重要意义。

关键词: 二花脸猪, 卵巢颗粒细胞, Linc-NORFA, 核心启动子, 转录调控

Abstract:

【Objective】 In our previous study, linc-NORFA has been proved as a candidate gene for sow fertility and participated in regulating follicular atresia and granulosa cell apoptosis. The aim of this study is to identify the core promoter of linc-NORFA and investigate its transcriptional regulation in Erhualian pig, so as to provide theoretical basis and new ideas for revealing the mechanism of linc-NORFA regulation to ovarian follicular atresia.【Method】 Ear samples of Euhualian pig were collected for genomic DNA extraction. PCR amplification and clone sequencing were used to obtain the 5’-flanking sequence of Erhualian pig linc-NORFA gene. Reporter vectors construction and luciferase activity assays were performed to identify the core promoter of linc-NORFA gene, and bioinformatic methods were conducted to analyze the characterization of linc-NORFA core promoter and the potential binding elements of transcription factors (TFs). In addition, pig FOXO1 gene eukaryotic expression vector was constructed and Western blot, qRT-PCR, and luciferase activity assays were performed to analyze the effects and regulatory mechanism of FOXO1 overexpression on the transcription of linc-NORFA gene. Besides, chromatin immunoprecipitation (ChIP) assay was performed to identify the interaction between FOXO1 and the core promoter of linc-NORFA in porcine granulosa cells (GCs).【Result】 A total of 1 734 bp 5’-flanking sequence of Erhualian pig linc-NORFA was obtained by PCR amplification and clone sequencing technology, which contained two potential CpG islands. Luciferase activity assay was performed and demonstrated that the core promoter of Erhualian pig linc-NORFA was located at -988 — -684 bp (TSS as +1). Multiple potential binding elements of several transcription factors (TFs) were identified within the core promoter of linc-NORFA using bioinformatic analyses, including ESR2, FOXO1, E2F1, BRCA1 and NFIC. In addition, results from ChIP assay proved that FOXO1 directly binds to the core promoter of linc-NORFA by acting as a transcription factor. Furthermore, It was proved that overexpression of FOXO1 could significantly down-regulate the activity of linc-NORFA core promoter (P<0.01), and also notably inhibited the expression level of linc-NORFA in porcine GCs (P<0.01).【Conclusion】 In this study, the core promoter of Erhualian pig linc-NORFA was identified, and FOXO1 acts as a transcription factor was proved, which significantly inhibited linc-NORFA transcription in porcine GCs through binding and further down-regulating the activity of its core promoter. These findings were of great significance for investigating the molecular mechanism of down-regulation of linc-NORFA during follicular atresia.

Key words: Erhualian pig, ovarian granulosa cells, linc-NORFA, core promoter, transcription regulation

杜星,曾强,刘禄,李琦琦,杨柳,潘增祥,李齐发. 二花脸猪linc-NORFA核心启动子鉴定与转录调控分析[J]. 中国农业科学, 2021, 54(15): 3331-3342.

DU Xing,ZENG Qiang,LIU Lu,LI QiQi,YANG Liu,PAN ZengXiang,LI QiFa. Identification of the Core Promoter of Linc-NORFA and Its Transcriptional Regulation in Erhualian Pig[J]. Scientia Agricultura Sinica, 2021, 54(15): 3331-3342.

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引物名称 Primer name	基因 Gene	引物序列 Sequence (5′-3′)	退火温度 Tm (℃)	大小 Size (bp)	用途 Usage
P1	linc-NORFA	F: TGGTCCAGGAGGGCAAGC	62.3	637	PCR扩增 PCR Amplification
P1	linc-NORFA	R: CCCCGTTTGCGTGCTTGT	62.3	637
P2	linc-NORFA	F: GGGTTTGTGAGTCCAAAGCAAG	61.9	796
P2	linc-NORFA	R: GGTTTCCACGTCCCTCGGTAT	61.9	796
P3	linc-NORFA	F: TTCCAGAGCCTCCAAAGTGA	58.1	730
P3	linc-NORFA	R: CCTTTGGGAGACCAGCACA	58.1	730
pNORFA-1F	linc-NORFA	CCAGCATCGCGTCGTCTTT	61.8	280	载体构建 Vector Construction
pNORFA-2F	linc-NORFA	TTCCAGAGCCTCCAAAGTGACT	58.8	613
pNORFA-3F	linc-NORFA	AGGCTGTGCCCAGCGCCTG	59.6	917
pNORFA-4F	linc-NORFA	GGGTTTGTGAGTCCAAAGCAA	60.2	1232
pNORFA-5F	linc-NORFA	GGTCGTCATGGAATATGCTCG	60.5	1771
pNORFA-R	linc-NORFA	CCTCCCAATACGATCTACTTCCTG	-	-
NORFA-RT	linc-NORFA	F: ATCCGTTTAGGTCCGTTAGAGC	60.7	117	qRT-PCR
NORFA-RT	linc-NORFA	R: GGAGGCTGAGTTTGCCACAA	60.7	117
FOXO1-RT	FOXO1	F: GGTCAAGAGCGTGCCCTACT	60.3	173
FOXO1-RT	FOXO1	R: TCCCACTCTTGCCTCCCTCT	60.3	173
GAPDH-RT	GAPDH	F: GGACTCATGACCACGGTCCAT	60.0	220
GAPDH-RT	GAPDH	R: TCAGATCCACAACCGACACGT	60.0	220
FBE-1	linc-NORFA	F:ACCTGTAATCCTGCTGGTGAGA	58.0	258	ChIP
FBE-1	linc-NORFA	R:CCACCCACAGGTCTTAATCCA	58.0	258
FBE-X	linc-NORFA	F:ATATGCTCGGTCCTGTGGC	60.0	328
FBE-X	linc-NORFA	R:CCACCTGTCAACCATCCCT	60.0	328