人PCAN1基因启动子的克隆及其上游调控区域的鉴定

山东大学学报(医学版)

人PCAN1基因启动子的克隆及其
上游调控区域的鉴定

刘闻闻¹，张菊²，关恒云²，张鹏举²，陈蔚文²，康鲁东²，
胡晓燕²，吴伟芳²，姜安丽²

（山东大学 1. 附属省立医院耳鼻喉头颈外科, 济南 250022；
2. 医学院生物化学与分子生物学研究所，济南 250012）

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-03-16 发布日期:2009-03-16
通讯作者: 姜安丽

Molecular cloning of the human PCAN1 promoter and identification of
its 5′ regulatory regions

LIU Wenwen¹, ZHANG Ju², GUAN Hengyun², ZHANG Pengju², CHEN Weiwen², KANG Ludong²,
HU Xiaoyan², WU Weifang², JIANG Anli²

(1. OtolaryngologyHead and Neck Surgery, Provincial Hospital Affiliated to Shandong University, Jinan 250022, China;
2. Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University， Jinan 250012, China)

Received:1900-01-01 Revised:1900-01-01 Online:2009-03-16 Published:2009-03-16
Contact: JIANG Anli

摘要/Abstract

摘要： 目的克隆人前列腺癌基因1（PCAN1）5′上游2.6?kb启动子片段，构建pGL3p2.6?kb载体, 测定其启动子活性，初步鉴定该片段内的DNA调控区域。方法采用PCR法从人基因组DNA中扩增PCAN1基因5′上游2.6?kb启动子片段，并构建到荧光素酶报道基因载体pGL3basic中，构成pGL3p2.6?kb；瞬时转染前列腺癌细胞LNCaP，通过双荧光素酶活性检验测定PCAN1启动子活性。使用Eraseasystem对pGL3p2.6kb载体中2.6?kb片段进行一系列5′侧翼缺失，产生12个缺失体，分别瞬时转染LNCaP细胞，通过检测荧光素酶的活性观察缺失突变对PCAN1启动子活性的影响。结果克隆的PCAN1 2.6?kb启动子片段经测序鉴定正确无误; pGL3p2.6?kb转染LNCaP细胞后，双荧光素酶活性测定结果显示2.6?kb片段具有明显的启动子活性；5′缺失突变分析显示，PCAN1基因上游-1?599?bp?至-1?541?bp、-347?bp至-84?bp的缺失，与2.6?kb?片段相比，启动子活性分别升高2.24倍和2.53倍，-1?541?bp?至-1?226?bp的缺失，与2.6?kb片段相比，启动子活性降低2.11倍。结论克隆的人PCAN1基因5′上游2.6?kb片段具有较强的启动子活性，PCAN1基因上游2.6?kb区域内分别存在2个负调控区和1个正调控区。

关键词: PCAN1基因, 启动区(遗传学), 基因缺失, 基因表达调控

Abstract:

To analyze the regulatory regions upstream of the human PCAN1 gene, a reporter plasmid containing of a 2.6?kb PCAN1 promoter was constructed by the PCR method, and DNA cisacting elements within this promoter were further identified. MethodsA 2.6?kb fragment upstream of the PCAN1 gene was amplified by PCR using human genomic DNA as a template, and it was then inserted into the upstream of the luciferase reporter gene in pGL3basic vector to generate pGL3p2.6kb. Its promoter activity was determined with dualluciferase reporter assay after it has been transfected into prostate cancer LNCaP cells. A series of 5′ deletion mutantluciferase reporter plasmids derived from pGL3p2.6kb were obtained via Eraseabase System. The mutant plasmids were transfected into the LNCaP cells and the promoter activities were determined by dualluciferase reporter assay. ResultsThe sequence of the 2.6?kb promoter fragment proved to be correct by DNA sequecing. Dualluciferase reporter assay showed that the 2.6?kb fragment presented a significant promoter activity with pRLTK into LNCaP cells. Analysis of the mutants showed that PCAN1 promoter activities increased 2.24fold and 2.53fold after the -1?599?bp to -1?541?bp and -347?bp to -84?bp within the promoter were deleted, while the promoter activity decreased 2.11fold after the -1?541?bp to -1?226?bp was deleted. ConclusionA cloned 2.6?kb fragment upstream of the PCAN1 gene presents strong promoter activity. With deletion analysis two negative and one positive regulatory region were identified within the promoter.

Key words: PCAN1 gene, Promoter regions (Genetics), Gene deletion, Gene expression

中图分类号:

Q344.14

刘闻闻1 ，张菊2 ，关恒云2 ，张鹏举2 ，陈蔚文2 ，康鲁东2 ，
胡晓燕2 ，吴伟芳2 ，姜安丽2
. 人PCAN1基因启动子的克隆及其
上游调控区域的鉴定[J]. 山东大学学报(医学版), 2009, 47(03): 4-7.

LIU Wenwen¹, ZHANG Ju², GUAN Hengyun², ZHANG Pengju², CHEN Weiwen², KANG Ludong²,
HU Xiaoyan², WU Weifang², JIANG Anli²

Molecular cloning of the human PCAN1 promoter and identification of
its 5′ regulatory regions

[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2009, 47(03): 4-7.

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