[1]余河玲,朱师良,何启牮,等.PDPK1 3′UTR双荧光素酶报告载体的构建及与gga-miR-148a-5p的靶向验证[J].江苏农业学报,2020,(01):147-151.[doi:doi:10.3969/j.issn.1000-4440.2020.01.020]
　YU He-ling,ZHU Shi-liang,HE Qi-jian,et al.Construction of PDPK1 3′UTR dual luciferase reporter vector and targeting verification with gga-miR-148a-5p[J].,2020,(01):147-151.[doi:doi:10.3969/j.issn.1000-4440.2020.01.020]
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PDPK1 3′UTR双荧光素酶报告载体的构建及与gga-miR-148a-5p的靶向验证()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2020年01期
页码:
147-151
栏目:
畜牧兽医·水产养殖
出版日期:
2020-02-29

文章信息/Info

Title:
Construction of PDPK1 3′UTR dual luciferase reporter vector and targeting verification with gga-miR-148a-5p
作者:
余河玲; 朱师良; 何启牮; 王彦
（四川农业大学畜禽遗传资源发掘与创新利用四川省重点实验室，四川成都611130）
Author(s):
YU He-ling; ZHU Shi-liang; HE Qi-jian; WANG Yan
(Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China)
关键词:
PDPK1; gga-miR-148a-5p; 禽白血病; 双荧光素酶报告基因检测系统
Keywords:
PDPK1; gga-miR-148a-5p; avian leukosis; dual luciferase reporter gene detection system
分类号:
Q783
DOI:
doi:10.3969/j.issn.1000-4440.2020.01.020
文献标志码:
A
摘要:
本试验通过构建PDPK1基因3′UTR双荧光素酶报告载体，并验证gga-miR-148a-5p与PDPK1的靶向关系。利用生物信息学软件预测gga-miR-148a-5p与PDPK1结合位点；利用PCR扩增PDPK1基因3′UTR序列，将其克隆到PGL3-CMV-LUC-MCS双荧光素酶报告基因载体中；将gga-miR-148a-5p及阴性对照分别与野生型gga-PDPK1-WT-3′UTR双荧光素酶报告质粒共转染至293T细胞中，双荧光素酶报告系统检测各组荧光素酶活性。Targetscan、miRbase数据库预测结果显示，gga-miR-148a-5p与PDPK1基因3′UTR存在互补结合位点；酶切及测序结果表明，双荧光素酶报告载体构建成功；荧光素酶活性试验结果表明，gga-miR-148a-5p mimics能够与PDPK1基因3′UTR结合并抑制荧光素酶活性。gga-miR-148a-5p能够与PDPK1靶向性结合，PDPK1是gga-miR-148a-5p的靶基因。
Abstract:
In this study, the PDPK1 gene 3′UTR dual luciferase reporter vector was established, and the targeting relationship between gga-miR-148a-5p and PDPK1 was verified. Bioinformatics software was used to predict the binding site of gga-miR-148a-5p and PDPK1. The 3′UTR sequence of PDPK1 gene was amplified by PCR and cloned into PGL3-CMV-LUC-MCS dual luciferase reporter vector. The gga-miR-148a-5p and negative control were cotransfected with wild-type gga-PDPK1-WT-3′UTR dual luciferase reporter plasmid into 293T cells, and the luciferase activity of each group was detected by dual luciferase reporter system. Prediction results from Targetscan and miRbase databases showed that there were complementary binding sites between gga-miR-148a-5p and the 3′UTR of PDPK1 gene. The enzyme digestion and sequencing results showed that the dual luciferase reporter vector was successfully constructed. The results of luciferase activity assay indicated that gga-miR-148a-5p mimics could bind to the 3′UTR of PDPK1 gene and inhibit luciferase activity. The gga-miR-148a-5p can bind to PDPK1, and PDPK1 is the target gene of gga-miR-148a-5p.

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备注/Memo

备注/Memo:
收稿日期：2019-07-08基金项目：国家自然科学基金项目（31601936）；四川省“十三五”育种攻关项目（2016NYZ0050）作者简介：余河玲（1992-），女，重庆云阳人，硕士研究生，主要从事家禽遗传育种与繁殖研究。 (E-mail)760963242@qq.com通讯作者：王彦，(E-mail)as519723614@163.com
更新日期/Last Update: 2020-03-13