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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p CAMBIA-rd29A-MsDREB1和p CAMBIA-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MsDREB1转基因大豆。对T_1至T_2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-q PCR)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MsDREB1基因在大豆耐旱基因工程中的应用提供方法。
The purpose of this study was to improve the drought tolerance of soybean (Glycine max) by using transgenic technology and to study the driving effect of two promoters of rd29A and CaMV-35S. Using the constructed transgenic vectors p CAMBIA-rd29A-MsDREB1 and p CAMBIA-35S-MsDREB1, the Medicago sativa gene DREB1 was introduced into the soybean variety Zhonghuang 13 by Agrobacterium tumefaciens-mediated method to obtain rd29A And CaMV-35S two types of promoters driven by MsDREB1 transgenic soybean. T_1 to T_2 generation of plants were analyzed by PCR and Southern blot. Nine and 12 transgenic soybean lines were screened, and two transgenic lines were randomly selected as the research object. Initial flowering under normal water conditions Soybean plant height and leaf area. Under 30 days of age, the differences of gene expression were analyzed by RT-qPCR under different drought stress conditions. The chlorophyll content, MDA content, relative water content and plant dry weight were measured. Department of rehydration after drought survival rate. The results showed that the expression of MsDREB1 was significantly different between the two promoters. MsDREB1 regulated by 35S promoter was overexpression but MsdREB1 regulated by promoter rd29A was lower under severe drought stress. The expression of rd29A : MsDREB1 expression is higher than 35S: MsDREB1 expression; MsDREB1 overexpression inhibits normal plant growth. The two promoters of each transgenic lines have a certain drought tolerance, but there are differences. MsDREB1 induced drought tolerance more effectively. Under moderate drought stress, the relative water content, chlorophyll content and dry weight per plant of MsDREB1 were significantly higher than that of MsDREB1 overexpression, whereas MDA content of MsDREB1 was significantly lower than that of MsDREB1 expression. The results indicate that MsDREB1 is involved in the regulation of drought in plants as a transcriptional regulator. This study provided a method for the application of MsDREB1 gene in soybean drought tolerant genetic engineering.