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.
作者
金晶
余昕健
靳锴
李婷婷
张晨
左其生
李碧春
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)
出处
《中国家禽》
北大核心
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)。
