鸡PERP1基因功能分析、核心启动子筛选及其转录因子预测

doi:10.16431/j.cnki.1671-7236.2023.09.001

摘要/Abstract

摘要： 【目的】研究TP53凋亡效应因子(PERP1)在卵泡发育中的调控作用及表达机制。【方法】以蛋鸡卵巢组织为材料,克隆蛋鸡PERP1基因CDS区全长序列,并构建PERP1基因过表达载体,通过转染鸡卵泡颗粒细胞,采用实时荧光定量PCR检测转染后PERP1基因,增殖凋亡相关基因BCL2、c-Myc,以及氧化应激相关基因SOD2、CAT的相对表达量。利用3个在线生物信息学分析软件分别预测鸡PERP1基因核心启动子区,并根据预测结果设计6个启动子缺失片段引物,以蛋鸡血液组织为材料,克隆PERP1基因5'-UTR的6个启动子缺失片段并构建重组质粒,并以荧光素酶报告基因试验验证启动子活性区域位置。采用4个转录因子在线预测软件进行启动子活性区转录因子的预测分析。【结果】本研究成功获得鸡PERP1基因的CDS区全长序列并成功构建了PERP1基因的过表达载体。PERP1基因过表达后,与对照组相比,抗凋亡基因BCL2和促增殖基因c-Myc表达量极显著下调(P＜0.01),抗氧化应激基因CAT和SOD2的相对表达量无显著差异(P＞0.05)。生物信息学软件预测和荧光素酶报告载体试验结果表明,PERP1基因核心启动子位于CDS区上游(―441/―71 bp)的位置。转录因子预测结果发现Sp1、ZEB1、Zic3、c-Jun、AP-2alpha、AP-1、NF-1、c/EBPalp、Adf-1、HNF-3、CACCC-bi、c-Myc和Smad4共13个候选转录因子。【结论】PERP1基因具有促进颗粒细胞凋亡的功能,调控该基因的表达对卵泡发育具有重要意义。

关键词: 鸡; PERP1基因; 核心启动子; 颗粒细胞; 转录因子

Abstract: 【Objective】 The purpose of this study was to investigate the regulatory role and expression mechanism of TP53 apoptotic effector factor (PERP1) in follicular development.【Method】 Using ovarian tissue of laying hens as material,the full length sequence of the CDS region of PERP1 gene in laying hens was cloned,and an overexpression vector of the PERP1 gene was constructed.The relative expression of PERP1 gene,proliferation and apoptosis related genes BCL2 and c-Myc,and oxidative stress related genes SOD2 and CAT after transfection were detected using Real-time quantitative PCR.Three online bioinformatics analysis software were used to predict the core promoter region of PERP1 gene in chicken,and six promoter deletion primers were designed according to the prediction results.Using blood tissues of laying hens as materials,six promoter deletion fragments of PERP1 gene 5 '-UTR were cloned and recombinant plasmids were constructed,and the location of the promoter active region was verified by Luciferase reporter gene test.Use four transcription factor online prediction software to predict and analyze transcription factors in the promoter active region.【Result】 This study successfully obtained the full length sequence of the CDS region of PERP1 gene in chicken and constructed an overexpression vector for PERP1 gene.After overexpression of PERP1 gene, compared with control group, the anti apoptotic gene BCL2 and proliferative gene c-Myc were significantly downregulated (P＜0.01),while the relative expression of antioxidant stress genes CAT and SOD2 were not significantly different (P＞0.05).The results of bioinformatics software prediction and Luciferase report vector test showed that the core promoter of PERP1 gene was located upstream of the CDS region (―441/―71 bp).The transcription factor prediction results revealed 13 candidate transcription factors,including Sp1,ZEB1,Zic3,c-Jun,AP-2alpha,AP-1,NF-1,c/EBPalp,Adf-1,HNF-3,CACCC-bi,c-Myc,and Smad4.【Conclusion】 The PERP1 gene had the function of promoting granulosa cell apoptosis,and regulating its expression was of great significance for follicular development.

Key words: chickens; PERP1 gene; core promoter; granulosa cell; transcription factors

中图分类号:

S831.2
Q78

陈林, 王家乡, 吴艳, 皮劲松, 张颖, 李成凤. 鸡PERP1基因功能分析、核心启动子筛选及其转录因子预测[J]. 中国畜牧兽医, 2023, 50(9): 3449-3458.

CHEN Lin, WANG Jiaxiang, WU Yan, PI Jinsong, ZHANG Ying, LI Chengfeng. Functional Analysis, Core Promoter Screening and Transcription Factors Prediction of PERP1 Gene in Chickens[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3449-3458.

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