[1]王潮岗,黄惠珠,孙海珊,等.一种莱茵衣藻启动子功能检测系统的构建[J].深圳大学学报理工版,2012,29(No.6(471-580)):534-540.[doi:10.3724/SP.J.1249.2012.06534]
　WANG Chao-gang,HUANG Hui-zhu,SUN Hai-shan,et al.Construction of Chlamydomonas reinhardtii system for analyzing the function of algal promoter[J].Journal of Shenzhen University Science and Engineering,2012,29(No.6(471-580)):534-540.[doi:10.3724/SP.J.1249.2012.06534]
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一种莱茵衣藻启动子功能检测系统的构建()
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《深圳大学学报理工版》[ISSN:1000-2618/CN:44-1401/N]

卷:
第29卷
期数:
2012年No.6(471-580)
页码:
534-540
栏目:
生物工程
出版日期:
2012-11-30

文章信息/Info

Title:
Construction of Chlamydomonas reinhardtii system for analyzing the function of algal promoter
文章编号:
20120612
作者:
王潮岗; 黄惠珠; 孙海珊; 胡章立; 雷安平
深圳大学生命科学学院，深圳市海洋生物资源与生态环境重点实验室，深圳 518060
Author(s):
WANG Chao-gang;  HUANG Hui-zhu;  SUN Hai-shan;  HU Zhang-li;  and LEI An-ping
Shenzhen Key Laboratory of Marine Biological Resources and Ecological Environment, College of Life Sciences, Shenzhen University, Shenzhen 518060, P.R.China
关键词:
基因工程; T载体; 莱茵衣藻; 启动子功能; 检测系统; 珠磨法;  克隆
Keywords:
genetic engineering;  T-vector;  Chlamydomonas reinhardtii;  promoter function;  analyzing system;  glass-bead method;  cloning
分类号:
Q 782
DOI:
10.3724/SP.J.1249.2012.06534
文献标志码:
A
摘要:
选用莱茵衣藻（Chlamydomonas reinhardtii）作为检测微藻启动子功能的生物系统，以pSP124质粒为基础，雨生红球藻β-胡萝卜素酮化酶基因bkt1的启动子片段（450 base pair, 记作450 bp）为间隔序列，引入两个Eam1105Ⅰ限制性内切酶位点，构建莱茵衣藻启动子功能检测T载体．将聚合酶链式反应(polymerase chain reaction，PCR)获得的1 986 bp的bkt1启动子片段直接克隆到T载体上，通过“珠磨法”转化到莱茵衣藻CC-849中，经Zeomycin筛选获得了TranB-0.45和TranBle转基因藻，而1 986 bp的bkt1启动子无转化子，原因可能是其含有负调控元件．PCR结果显示，ble可稳定存在于转基因藻中，bkt1启动子的450 bp片段具有启动子活性，能正确表达BLE蛋白．该研究表明，莱茵衣藻和pB-0.45T载体所组成的检测系统可用于藻类启动子研究，为启动子功能的研究提供了一条新途径．
Abstract:
Chlamydomonas reinhardtii was employed as a bio-system for analyzing the function of algal promoter in this study. The C.reinhardtii T-vector based on pSP124 plasmid was constructed using 450 bp sequences of β- carotene ketolase gene (bkt1) promoter as insert fragment. Two Eam1105Ⅰ restriction sites were introduced into this T-vector. 1 986 bp sequences of bkt1 promoter was obtained by PCR and cloned into the C.reinhardtii T-vector directly. By “glass-bead method”, transformants of TranB-0.45 and TranBle were generated from TAP containing 10 μg/mL Zeomycin. However, none of TranB-2 were observed due to some negative regulatory elements in 1 986 bp-bkt1 promoter. Results of PCR confirmed that ble was integrated into the genome DNA of C.reinhardtii. The 450 bp sequences of bkt1 promoter were able to express BLE in transgenic algae, which verified that it owned promoter ability in C.reinhardtii. These results indicate that the C.reinhardtii and pB-0.45T system for analyzing promoter function is workable. It is a new way for studying algal promoters.

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备注/Memo

备注/Memo:
Received：2012-08-02；Accepted：2012-10-29
Foundation：National Natural Science Foundation of China (31000162, 31070323)
Corresponding author：Professor HU Zhang-li. E-mail: huzl@szu.edu.cn
Citation：WANG Chao-gang，HUANG Hui-zhu，SUN Hai-shan，et al. Construction of Chlamydomonas reinhardtii system for analyzing the function of algal promoter［J］. Journal of Shenzhen University Science and Engineering, 2012, 29(6): 534-540.（in Chinese）
基金项目：国家自然科学基金资助项目(31000162，31070323)
作者简介：王潮岗( 1978-) ，男( 汉族) ，广东省揭阳市人，深圳大学讲师、博士. E-mail: ?charleswangcn@163.com
引文：王潮岗，黄惠珠，孙海珊，等. 一种莱茵衣藻启动子功能检测系统的构建［J］. 深圳大学学报理工版，2012，29（6）：534-540.
更新日期/Last Update: 2012-11-30