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

Abstract

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

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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References 44

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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

Identification of the Core Promoter of Linc-NORFA and Its Transcriptional Regulation in Erhualian Pig

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