《植物生理学报》 2010, 46(7): 724-730

麻疯树MIPS 基因启动子的分离及在烟草原生质体中瞬时表达活性分析

苟春宝, 王勇, 喻川, 陈放, 魏炜*

四川大学生命科学学院, 生物资源与生态环境教育部重点实验室, 成都610064

通信作者：魏炜；E-mail: wwxfbxw@gmail.com；Tel: 028-85410 672

摘　要：

根据麻疯树 MIPS 基因序列, 设计特异性的巢式引物, 运用 TAIL-PCR 法两次步移得到 MIPS 基因 5' 端侧翼序列, 序列 分析显示含有多个胁迫应答相关元件, 如 ABRE、HSE 等。以该序列为基础, PCR 扩增得到 5 个 5' 端不同长度的缺失片段, 分别插入 pBI221 载体置换 CaMV 35S 启动子, 构建的表达载体在 PEG 介导下转入烟草叶片原生质体进行瞬时表达, 检测 GUS 报告基因的活性。经 GUS 活性荧光定量检测发现, 分离到的 MIPS 基因侧翼序列 5' 端不同缺失片段都能启动 GUS 报 告基因表达, 启动活性最高的是 WQ1 区(−565 bp), 核心区位于 −565~−449 bp。在 100 μmol•L-1 ABA 诱导下启动活性增强, 但不同区段的增长幅度不同。WQ1 区增长幅度最大, 比未处理时提高 41.4%。

关键词：麻疯树; MIPS基因; 启动子; 原生质体; 瞬时表达

收稿：2010-01-19   修定：2010-06-18

资助：“ 十一五” 科技支撑项目(2006BAD07A04)和四川省“ 十一五” 支撑项目(2007BAD50B00)

Isolation of MIPS Gene Promoter from Jatropha curcas L. and Activity Analysis of Transient Expression in Tobacco Protoplast

GOU Chun-Bao, WANG Yong, YU Chuan, CHEN Fang, WEI Wei*

Key Laboratory of Bio-resource and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China

Corresponding author: WEI Wei; E-mail: wwxfbxw@gmail.com; Tel: 028-85410 672

Abstract:

Using nested primers specific for the sequences of Jatropha curcas MIPS gene, the MIPS gene 5'-flanking sequences were obtained by TAIL-PCR. Some stress response elements were found by sequence analysis, such as ABA-responsive element (ABRE) and heat stress responsive element (HSE). Five different 5'-end deletion fragments of the MIPS gene 5'-flanking sequences were amplified by PCR, and they were inserted into the plant transient expression vector pBI221 to replace CaMV 35S promoter respectively. The expression vectors were transferred into tobacco leaf protoplasts by PEG-mediated transient expression and the different promoter activities were quantitatively estimated using GUS report gene. The results demonstrated that the isolated 5'-end different deletion fragments of MIPS gene could initiate GUS reporter gene expression in a tobacco leaf protoplast transient expression system. The GUS activity of WQ1 area (−565 bp) was the highest and the core area was located between −565 bp and −449 bp. The activity was increased by ABA (100 μmol·L^-1) treatment, but the increase in GUS activity of different segments was not the same. GUS activity was increased by 41.4% in WQ1 area, when compared with the control.

Key words: Jatropha curcas; MIPS gene; promoter; protoplast; transient expression