鸡PERP1基因功能分析、核心启动子筛选及其转录因子预测  

Functional Analysis, Core Promoter Screening and Transcription FactorsPrediction of PERP 1 Gene in Chickens

作　　者：陈林 王家乡[1] 吴艳[2] 皮劲松[2] 张颖 李成凤 CHEN Lin;WANG Jiaxiang;WU Yan;PI Jinsong;ZHANG Ying;LI Chengfeng(School of Animal Science,Yangtze University,Jingzhou 434025,China;Animal Husbandry and Veterinary Research Institute,Hubei Academy of Agricultural Sciences,Hubei Provincial Key Laboratory of Animal Embryo Engineering and Molecular Breeding,Wuhan 430064,China;Hubei Shendan Health Food Co.,Ltd.,Wuhan 432600,China)

机构地区：[1]长江大学动物科学学院,荆州434025 [2]湖北省农业科学院畜牧兽医研究所,动物胚胎工程及分子育种湖北省重点实验室,武汉430064 [3]湖北神丹健康食品有限公司,武汉432600

出　　处：《中国畜牧兽医》2023年第9期3449-3458,共10页China Animal Husbandry & Veterinary Medicine

基　　金：湖北省科技重大专项(2020ABA016);农业农村部和财政部:现代农业产业技术体系建设专项资金(CRAS-40-s15)。

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。【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 PERP 1 gene in laying hens was cloned,and an overexpression vector of the PERP 1 gene was constructed.The relative expression of PERP 1 gene,proliferation and apoptosis related genes BCL 2 and c-Myc,and oxidative stress related genes SOD 2 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 PERP 1 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 PERP 1 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 PERP 1 gene in chicken and constructed an overexpression vector for PERP 1 gene.After overexpression of PERP 1 gene,compared with control group,the anti apoptotic gene BCL 2 and proliferative gene c-Myc were significantly downregulated(P<0.01),while the relative expression of antioxidant stress genes CAT and SOD 2 were not significantly different(P>0.05).The results of bioinformatics software prediction and Luciferase report vector test showed that the core promoter of PERP 1 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 PERP 1 gene had the function of pro

关 键 词：鸡 PERP1基因 核心启动子 颗粒细胞 转录因子 

分 类 号：S831.2[农业科学—畜牧学] Q78[农业科学—畜牧兽医]