鸡lncRNA-CEPT8启动子验证及活性核心区域筛选  

Promoter Verification and Active Core Region Screening of lncRNA-CEPT8 in Chicken

作　　者：金晶[1,2] 余昕健 靳锴 李婷婷 张晨[1,2] 左其生 李碧春[1,2] JIN Jing;YU Xinjian;JIN Kai;LI Tingting;ZHANG Chen;ZUO Qisheng;LI Bichun(College of Animal Science and Technology,Yangzhou University,Yangzhou,Jiangsu 225009;Jiangsu Key Laboratory of Animal Breeding Reproduction and Molecular Design,Yangzhou,Jiangsu 225009;Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming,Yunnan 650223)

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009 [2]江苏省动物遗传繁育与分子设计重点实验室,江苏扬州225009 [3]中国科学院昆明动物研究所,云南昆明650223

出　　处：《中国家禽》2020年第6期15-20,共6页China Poultry

基　　金：江苏省科技计划(青年基金)(BK20180918);江苏省高校自然科学研究项目(18KJB230008);江苏省研究生科研与实践创新计划项目(KYCX19_2114);国家重点研发计划(2017YFE0108000);国家自然科学基金项目(31872341)。

摘　　要：研究旨在筛选鸡lncRNA-CEPT8启动子核心区域并验证其启动活性。采用qRT-PCR检测lncRNA-CEPT8在鸡不同组织中表达情况。生物信息学预测lncRNA-CEPT8启动子区域,PCR扩增启动子并构建pEGFP-lncCEPT8重组载体,转染DF-1细胞后48 h进行荧光观察。再构建启动子系列缺失载体(pGL3-p1、pGL3-p2、pGL3-p3、pGL3-p4、pGL3-p5),分别转染DF-1细胞后48 h检测双荧光素酶活性,分析缺失载体的启动活性。结果表明,lncRNA-CEPT8在鸡生殖嵴中特异性高表达,pEGFP-lncCEPT8重组载体转染DF-1细胞后有绿色荧光。缺失载体pGL3-p4与pGL3-p3相比,活性极显著下降(P<0.01)。研究明确了lncRNA-CEPT8上游-1174^-1 bp处为启动子,且-627^-429bp是启动子核心区域,为进一步研究其表达调控机制提供理论依据。In order to screen core region of the promoter of lncRNA-CEPT8 and verify its promoter activity,qRT-PCR was used to detect the expression of lncRNA-CEPT8 in different tissues of chickens.lncRNA-CEPT8 promoter region was predicted by bioinformatic tools.The promoter segment was amplified by PCR and a recombination vector named pEGFP-lncCEPT8 was constructed.This vector was transfected into DF-1 cells to observe green fluorescence after 48 h.Then a series of deletion mutation vectors(pGL3-p1,pGL3-p2,pGL3-p3,pGL3-p4,pGL3-p5)was transfected into DF-1 cell to test dual-luciferase activity to analyze the activity of the promoter segments after 48 h.The results showed that lncRNA-CEPT8 was highly expressed in genital ridge.Green fluorescence could be detected in DF-1 cells after transfecting the pEGFP-lncCEPT8.Compared with pGL3-p3,the promoter activity of pGL3-p4 was much lower(P<0.01).The study confirmed that the upstream of lncRNA-CEPT8 ranged from-1174 bp to-1 bp was the promoter,and-627 bp to-429 bp was the core region of the promoter.This conclusion laid the foundation for further study the expression mechanism of this lncRNA.

关 键 词：鸡 启动子 lncRNA 核心区域 

分 类 号：S831.2[农业科学—畜牧学]