烟草化学诱导表达系统的建立  

Establishment of A Chemical-inducible Gene Expression System in Tobacco

作　　者：代丽娟[1] 郑唐春[1] 刘彩霞[1] 刘轶[1] 由香玲[2] 曲冠证[1] 

机构地区：[1]东北林业大学林木遗传育种国家重点实验室,哈尔滨150040 [2]东北林业大学生命科学学院,哈尔滨150040

出　　处：《植物研究》2016年第6期917-924,共8页Bulletin of Botanical Research

基　　金：教育部新世纪优秀人才支持计划项目(No.NCET-12-0808);国家自然科学基金项目(No.31370661)资助~~

摘　　要：化学诱导表达系统对植物功能基因的研究及植物基因工程应用有重要意义。XVE是以雌激素为基础的用于诱导转基因植物中目的基因表达的一种系统,能够在可调控方式下诱导基因的表达。目前,以烟草为遗传转化材料进行XVE系统研究的详细报道还未见发表。本文以烟草作为研究对象,利用PCR技术从本实验保存的质粒pROKII-GFP中扩增出GFP基因。构建雌激素诱导型植物表达载体(p ER8-GFP)并利用农杆菌介导的叶盘法将外源基因导入野生型烟草中;经潮霉素抗性筛选出转化植株后,用PCR鉴定出阳性转化植株,将阳性转化植株利用不同浓度和不同时间的雌激素进行处理,并结合定量PCR和Night SHADE植物活体成像系统对转基因植株的表达水平进行检测。结果表明诱导p ER8-GFP载体在烟草中表达的最适雌激素浓度为25μmol·L^(-1),最适时间为48 h;同时在烟草胚轴与根尖细胞中检测到荧光信号,表明XVE化学诱导系统在烟草中也可以高效、严格的依赖雌激素的诱导来控制目的基因表达。本研究将为烟草相关的化学诱导表达研究提供技术支持与解决方案。Chemical-inducible expression system is a powerful tool for plant functional genomics and plant genetic engineering applications. XVE is a chemical-inducible expression system using estrogen for chemical induction. The detailed report about XVE using tobacco as plant material was not available yet. In this study,GFP was amplified from the plasmid pROKII-GFP that was preserved in this experiment by PCR. Estrogen inducible plant expression vector（ p ER8-GFP） was constructed and transformed into wild-type tobacco by Agrobacterium-mediated leaf disc transformation. The positive transformants were screened with hygromycin followed by a further PCR identification. The transformants were induced with different concentrations and different times of estrogen. The expression of GFP in transgenic tobacco was tested by using qRT-PCR and Night SHADE plants in vivo imaging system,and the results showed that the best suitable concentration of estrogen which induced the expression of p ER8-GFP in tobacco was 25 μmol·L^（-1）,and the best suitable period was 48 h. At the same time,the GFP gene appeared in the hypocotyl and root tip cells of transgenic tobacco. It showed that the XVE chemical induction system in tobacco could also be used to control the expression of the target gene efficiently by the induction of estrogen. Our study will contribute to the XVE study of tobacco in future.

关 键 词：雌激素诱导 基因表达 XVE 烟草 

分 类 号：Q943[生物学—植物学]