《植物生理学报》 2017, 53(8): 1479-1488

过表达苜蓿MsDREB1基因大豆耐旱性分析

李大红, 郑文娜^*, 蒋炳伸, 李鸿雁

黄淮学院生物与食品工程学院, 河南驻马店463000

通信作者：郑文娜；E-mail: 394530162@qq.com

摘　要：

本研究目的是利用转基因技术改良大豆(Glycine max)的耐旱性, 并研究rd29A和CaMV-35S两类启动子的驱动效果。利用构建的转基因载体pCAMBIA-rd29A-MsDREB1和pCAMBIA-35S-MsDREB1, 通过农杆菌(Agrobacterium tumefaciens)介导法, 将苜蓿(Medicago sativa)基因DREB1导入大豆品种‘中黄13号’, 获得rd29A和CaMV-35S两类启动子驱动的Ms-DREB1转基因大豆。对T₁至T₂代植株进行PCR、Southern blot分析, 分别筛选到9和12个转基因大豆株系, 各随机选择两个转基因株系作为研究对象。正常水分状态下初花期统计大豆株高及叶面积。苗龄30 d的植株在不同干旱胁迫条件下, 用逆转录定量PCR (RT-qPCR)分析基因表达差异, 测定叶绿素含量、丙二醛含量、相对含水量及植株干重, 并分析各株系干旱后复水的成活率。结果表明, 两种启动子对MsDREB1表达的调控存在明显差异, 在非胁迫下35S启动子调控的MsDREB1为超量表达, 而rd29A启动子调控的MsDREB1表达量较低; 在严重干旱胁迫下, rd29A:MsDREB1表达量高于35S:MsDREB1表达量; MsDREB1超量表达抑制植株正常生长。两种启动子各转基因株系均有一定耐旱能力, 但存在差异。MsDREB1诱导表达耐旱性效果更明显, 在中度干旱胁迫下, 其植株相对含水量、叶绿素含量、单株干重均显著高于MsDREB1超量表达, 而丙二醛含量显著低于MsDREB1超量表达。结果说明MsDREB1作为转录调节因子参与了植物的干旱调节。该研究为Ms-DREB1基因在大豆耐旱基因工程中的应用提供方法。

关键词：大豆; MsDREB1; rd29A启动子; 耐旱性

收稿：2017-06-11   修定：2017-08-10

资助：河南省科技发展计划项目(112300410042)。

Drought tolerance analysis of transgenic soybean with overexpressed MsDREB1 gene from alfalfa

LI Da-Hong, ZHENG Wen-Na^*, JIANG Bing-Shen, LI Hong-Yan

School of Biology and Food Engineering, Huanghuai University, Zhumadian, Henan 463000, China

Corresponding author: ZHENG Wen-Na; E-mail: 394530162@qq.com

Abstract:

The purpose of this study was using transgenic technique to improve drought resistance of soybean (Glycine max) and also the driving effects of two kinds of promoters, rd29A and CaMV-35S. By using the transgenic vectors pCAMBIA-rd29A-MsDREB1 and pCAMBIA-35S-MsDREB1 into soybean through Agrobacterium tumefaciens-mediated transformation, the transgenic soybeans drived by rd29A and CaMV-35S were produced. By analyzing T₁ to T₂ generations plants by PCR and Southern blot, 9 and 12 transgenic soybean lines were selected, respectively, and then 2 transgenic soybean lines from each generation were randomly chosen as the research object. The height and area of soybean leaves statistics was couducted under well-watered situation at the beginning of flowering period. By analyzing the differences of 30 d old seedlings in gene expression under different drought stress conditions by reverse transcription PCR (RT-PCR), the contents of chlorophyll, malondialdehyde and relative water and dry weight of the plants were measured and the survival rate of every line was calculated after rewatering. The results show that there were significant differences in the regulation of MsDREB1 expression between the two promoters. Under non-stress condition, MsDREB1 gene was overexpressed under the regulation of CaMV-35S promoter, but the expression was lower under the regulation of rd29A promoter. Under severe drought-stress condition, the expression level of rd29A:MsDREB1 was higher than that of 35S:MsDREB1. The overexpression of MsDREB1 inhibited the normal growth of plants. Both promoter-drived transgenic lines had some abilities of drought tolerance, but there were also some differences between themselves. The effect of drought resistance of rd29A:MsDREB1 was more obvious. Its relative water and chlorophyll contents and dry weight were significantly higher than those of 35S:MsDREB1, while the content of malondialdehyde was significantly lower than the overexpression of 35S:MsDREB1. The aim of this study is to obtain transgenic soybean lines with drought tolerance by comparing the drought tolerance of transgenic soybean with MsDREB1 gene regulated by different promoters, and to provide an effective method of application of drought-stress genetic engineering with MsDREB1 genes in soybean.

Key words: soybean; MsDREB1; rd29A promoter; drought tolerance