大豆发状根乙醇诱导表达系统的建立及验证  

Construction and Verification of Ethanol Inducible Expression System in Soybean Hairy Root

作　　者：洪冰杰 许鑫[2] 侯文胜[2] 于丽杰[1] 韩天富 HONG Bingjie;XU Xin;HOU Wensheng;YU Lijie;HAN Tianfu(College of Life Science and Technology, Harbin Normal University, Harbin, Heilongjiang 150025;Key Laboratory of Soybean Biology (Beijing), Ministry of Agriculture/Institute of Crop Sciences,Chinese Academy of Agricultural Sciences, Beijing 100081)

机构地区：[1]哈尔滨师范大学生命科学与技术学院,黑龙江哈尔滨150025 [2]中国农业科学院作物科学研究所/农业部北京大豆生物学重点实验室,北京100081

出　　处：《核农学报》2019年第2期217-225,共9页Journal of Nuclear Agricultural Sciences

基　　金：国家重点研发计划(2017YFD0101400);国家现代农业产业技术体系建设专项(CARS-04)

摘　　要：为在大豆中建立目的基因表达可控的化学诱导表达系统,本研究构建了基于乙醇诱导表达系统(ALCA switch)的GUS和GmFT2a表达载体,利用发状根农杆菌介导的根系转化体系,分别获得转ALCA-mini35S-GUS和ALCA-mini35S-GmFT2a的发状根,通过检测GUS的表达情况和GUS酶活性高低评定该系统在大豆发状根中的诱导效率,并通过检测GmFT2a表达水平加以验证。结果表明,在非诱导条件下,ALCA-mini35S-GUS发状根中未见GUS表达,而在培养基中添加乙醇后组织化学染色效果明显,说明GUS基因表达。乙醇诱导表达的效率与乙醇浓度有关,当乙醇浓度为0.05%、处理60 h时GUS相对表达量达到最高值,处理72 h时GUS酶活性最高;0.05%乙醇处理72 h时,ALCA-mini35S-GmFT2a发状根中GmFT2a相对表达量平均值约为诱导前的150倍,个别样本可达初始值的400倍。综上所述,乙醇诱导表达系统可以在大豆发状根中发挥作用,该系统不仅可用于大豆基因功能研究,而且可为培育外源基因表达可控的转基因大豆品种提供新的技术手段。To establish a chemically inducible gene expression system in soybean, two ethanol inducible expression system(ALCA switch)-based transforming vectors harboring GUS and GmFT2a were constructed, respectively. And transformed into soybean hairy root. Then, the efficiency of induction in ALCA switch-based system was evaluated by measuring the transcriptional level of GUS and GUS enzymatic activity, and verified by testing the expression level of GmFT2a. The results demonstrated that under non-ethanol induction treatment, the expression level of GUS was undetectable in the hairy root transformed with ALCA-mini35S-GUS construct. However, the expression of GUS was observed by using histo-chemical staining after ethanol induction. The gene expression level in ALCA switch system was dependent on ethanol concentration. The highest transcriptional level of GUS gene was obtained by using 0.05% ethanol concentration with 60 hours, and enzymatic activity was the highest after 72 hours of ethanol induction. After 72 hours of ethanol induction(0.05% concentration), the average expression level of GmFT2a in hairy root with ALCA-mini35 S construct was increased 150 times than the non-induction treatment, and the maximum level in the extremely-expressed event was even increased 400 times than the control. This study demonstrated that ALCA switch system was functional in soybean hairy root, and this system constructed can not only be used in characterization of gene function, but also provides a novel tool for generating transgenic soybean cultivars.

关 键 词：大豆 乙醇诱导表达系统 GmFT2a 表达调控 

分 类 号：S565.1[农业科学—作物学]