《植物生理学报》 2014, 50(11): 1675-1682

水曲柳节律基因LHY启动子的克隆和功能分析

王璇², 曾凡锁^1,2, 詹亚光^1,2,*, 何之龙²

东北林业大学¹林木遗传育种国家重点实验室, ²生命科学学院, 哈尔滨150040

通信作者：詹亚光；E-mail: yaguangzhan@126.com；Tel: 0451-82191752

摘　要：

以水曲柳基因组DNA为模板, 用SiteFinding-PCR法扩增得到节律基因LHY (late elongated hypocotyl)启动子序列, 长度为1 360 bp。PLACE启动子预测工具分析表明, 序列中含有转录必备的TATA box、CAAT box以及一些非生物胁迫和激素响应元件等。构建植物GFP瞬时表达载体pPXGFP-P-LHY, 农杆菌介导转化烟草叶片和白桦悬浮细胞, GFP检测结果表明, LHY启动子能够启动GFP基因在烟草和白桦细胞中表达, 且对非生物胁迫(低温、高温、盐)产生响应; 构建植物GUS报告基因整合表达载体pPCXGUS-P-LHY, 农杆菌介导法瞬时转化烟草, GUS染色结果表明, LHY启动子的活性具有不同程度的时空特性。

关键词：LHY; 启动子; 克隆; 瞬时表达

收稿：2014-08-05   修定：2014-09-29

资助：国家科技支撑计划项目(2012BAD01B0503)和国家自然科学基金(31270697)。

Cloning and Functional Analysis of the Circadian Gene LHY Promoter in Fraxinus mandshurica

WANG Xuan², ZENG Fan-Suo^1,2, ZHAN Ya-Guang^1,2,*, HE Zhi-Long²

¹State Key Laboratory of Tree Genetics and Breeding, ²College of Life Sciences, Northeast Forestry University, Harbin 150040, China

Corresponding author: ZHAN Ya-Guang; E-mail: yaguangzhan@126.com; Tel: 0451-82191752

Abstract:

The LHY promoter fragment (1 360 bp) was isolated and identified from the genomic DNA of Fraxinus mandshurica using the method of SiteFinding-PCR. Promoter sequence was analysed by PLACE, and the result showed that this fragment contained TATA box and CAAT box, as well as some elements which can respond to abiotic stress and hormones. Plant transient expression vector with report gene GFP (named as pPXGFP-P-LHY) was constructed, and then was transformed into Nicotiana tabacum leaves and Betula platyphylla cells through the Agrobacterium infection process. The result showed that the promoter of LHY could activate the expression of GFP gene in N. tabacum leaves and B. platyphylla cells, and this activation could respond to abiotic stress, such as cold, heat and salinity. Furthermore, another plant expression vector with report gene GUS (named as pPCXGUS-P-LHY) was constructed too, and after transformation to N. tabacum leaves by Agrobacterium infection, the activity of LHY promoter showed different in stem and leaf vain tissue.

Key words: LHY; promoter; cloning; transient expression