猪CYP3A29基因核心启动子鉴定及转录调控分析

doi:10.11843/j.issn.0366-6964.2025.03.016

摘要/Abstract

摘要：

旨在鉴定猪CYP3A29基因核心启动子及相应的转录调控因子，分析转录因子对CYP3A29启动子活性的调控。本研究以3头健康的大白母猪(30 kg)为试验材料，利用PCR和Western blot检测CYP3A29基因在猪各组织(心、肝、脾、肺、肾、小肠、肌肉)中的表达分布；构建不同片段长度的CYP3A29基因启动子双荧光素酶报告载体，转染293T和AML12细胞系，检测荧光素酶活性，确定CYP3A29基因的核心启动子区域；利用Animal TFDB网站分析CYP3A29核心启动子区域可能存在的转录调控因子，针对核心启动子区域构建分段缺失双荧光素酶报告载体，检测荧光素酶活性大小，确定转录因子结合位点；构建转录因子结合突变位点的双荧光素酶报告载体和转录因子shRNA载体，探讨转录因子对CYP3A29核心启动子的调控作用。结果显示，CYP3A29基因在猪肝脏中表达量最高；CYP3A29启动子4个不同检测区域(-2 026~+62 bp、-1 526~+62 bp、-1 026~+62 bp和-528~+62 bp)中-528~+62 bp活性最高，为CYP3A29核心启动子区；CYP3A29启动子-528~-448 bp区域负向调控核心启动子活性，且含有潜在的转录因子RUNX1结合位点；突变RUNX1结合位点可显著降低-528~+62 bp启动子的荧光素酶活性，而干扰RUNX1基因则显著升高-528~+62 bp野生型启动子的荧光素酶活性，但对-528~+62 bp突变型启动子的荧光素酶活性无显著影响，提示RUNX1转录因子可负向调控CYP3A29基因核心启动子活性。本研究结果为进一步解析猪CYP3A29基因的转录调控机制奠定了基础。

关键词: 猪, CYP3A29, 核心启动子, RUNX1, 转录调控

Abstract:

The aim of this study was to identify the core promoter activity of porcine CYP3A29 gene and the corresponding transcriptional regulatory factors, and to analyze the regulation of CYP3A29 promoter activity by transcription factors. Three healthy Yorkshire sows (30 kg) were used as experimental materials to detect the expression and distribution of CYP3A29 gene in pig tissues (heart, liver, spleen, lung, kidney, small intestine and muscle) by PCR and Western blot. The dual luciferase reporter vectors of CYP3A29 gene promoter with different fragment lengths were constructed by PCR amplification and were transfected into 293T and AML12 cell lines. The core promoter region of CYP3A29 gene was determined by detecting luciferase activity. The animal TFDB website was used to analyze the possible transcription factor binding sites in the core promoter region of CYP3A29 gene and segmented deletion dual-luciferase reporter vectors of the core promoter region were constructed. The binding site region of the transcription factor was further determined by detecting the luciferase activity. The dual-luciferase reporter vectors with transcription factor binding mutation sites and shRNA vector for transcription factor were constructed, which were used to explore the role of transcription factors in the regulation of the CYP3A29 core promoter. The results showed that the expression of CYP3A29 gene was the highest in pig liver. The -528- +62 bp region of CYP3A29 promoter had the highest activity among the 4 different regions (-2 026-+62 bp, -1 526-+62 bp, -1 026- +62 bp and -528- +62 bp), and was considered to be the core promoter region. The -528- -448 bp region of CYP3A29 promoter negatively regulated the core promoter activity and contained a potential transcription factor binding site of RUNX1. The mutant RUNX1 binding site significantly reduced the luciferase activity of the -528- +62 bp promoter, while the interference of the RUNX1 gene significantly increased the luciferase activity of the -528- +62 bp wild-type promoter, but had no significant effect on the luciferase activity of the -528- +62 bp mutant promoter, indicating that RUNX1 negatively regulated the core promoter activity of the CYP3A29 gene. The results of this study laid a foundation for further analysis of the transcriptional regulation mechanism of CYP3A29 gene.

Key words: porcine, CYP3A29, core promoter, RUNX1, transcription regulation

中图分类号:

S828.2

王红, 赵为民, 程金花, 李惠侠, 方晓敏. 猪CYP3A29基因核心启动子鉴定及转录调控分析[J]. 畜牧兽医学报, 2025, 56(3): 1147-1158.

WANG Hong, ZHAO Weimin, CHENG Jinhua, LI Huixia, FANG Xiaomin. Identification and Transcriptional Regulation Analysis of the Core Promoter of Porcine CYP3A29 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1147-1158.

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引物Primer	引物序列(5′→3′)Primer sequence	片段长度/bp Length
PGL-CYP3A29(-2 026~+62)-F1	GCTG$\underline{{\rm{GCTAGC}}}$ TCTACTTCATGGTGCCCAGAG	2 088
PGL-CYP3A29(-1 526~+62)-F2	GCTG$\underline{{\rm{GCTAGC}}}$GAGCTGGGACTCACTGCATG	1 588
PGL-CYP3A29(-1 026~+62)-F3	GCTG$\underline{{\rm{GCTAGC}}}$TTCCTGCGTCACACCCTTCA	1 088
PGL-CYP3A29(-528~+62)-F4	GCTC$\underline{{\rm{GCTAGC}}}$AATTGTGCTCAGGCGGATAG	590
PGL-CYP3A29(-448~+62)-F5	GCTC$\underline{{\rm{GCTAGC}}}$AAGATAGACTATTCTTTCTGAGC	510
PGL-CYP3A29(-289~+62)-F6	GCTC$\underline{{\rm{GCTAGC}}}$GGAGACATGGCATTTTATAGG	351
PGL-CYP3A29(-96~+62)-F7	GCTC$\underline{{\rm{GCTAGC}}}$TGGTGTTTTTTCACTGGCTGC	158
PGL-CYP3A29-promoter-R	CCCAAGCTTGCCACTGTCCTCGTGATTCT
CYP3A29	F: ACATCTTTGGGGCCTACAGC R: AGATCGGGGTGAGGAATGGA	177
HPRT(pig)	F: TGACCAGTCAACGGGCGATA R: CAACACTTCGAGGGGTCCTT	197
HPRT(mouse)	F: CCATCACATTGTGGCCCTCT R: TTATGTCCCCCGTTGACTGA	167
RUNX1	F: TGGCAGGCAACGATGAAAAC R: GCAACTTGTGGCGGATTTGT	163
Spi1	F: GAACCACTTCACAGAGCTGC R: GTCATCTTCTTGCGGTTGCC	474
Spib	F: CAGAGGACTTCACCAGCCAG R: GAGGAGAACTGGAAGACGCC	232

引物Primer	引物序列(5′→3′)Primer sequence
shRUNX1-F	CACC$\underline{{\rm{GCAGAACTGAGAAATGCTACC}}}$CTCGAG$\underline{{\rm{GGTAGCATTTCTCAGTTCTG}}}$CTTTTTT
shRUNX1-R	AAACAAAAAA$\underline{{\rm{GCAGAACTGAGAAATGCTACC}}}$CTCGAG$\underline{{\rm{GGTAGCATTTCTCAGTTCTGC}}}$
shNC-F	CACCG$\underline{{\rm{TTCTCCGAACGTGTCACGT}}}$CTCGAG$\underline{{\rm{ACGTGACACGTTCGGAGAA}}}$CTTTTTT
shNC-R	AAACAAAAAAG$\underline{{\rm{TTCTCCGAACGTGTCACGT}}}$CTCGAG$\underline{{\rm{ACGTGACACGTTCGGAGAA}}}$C

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