盐地碱蓬DREB同源基因SsDREB的功能研究
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江苏省盐土生物资源研究重点实验室开放课题(JKLBS2012004)


Functional Study of a DREB homologous gene SsDREB from Suaeda salsa L.
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    摘要:

    SsDREB是从盐地碱蓬克隆的一个DREB2 类转录因子,其表达受高盐和干旱诱导,而对ABA 和低温处理反应不明显。本文将SsDREB与绿色荧光蛋白(GFP)基因构建融合表达载体并在洋葱表皮细胞中进行瞬时表达,研究结果表明SsDREB编码蛋白定位在细胞的细胞核中;酵母转录激活实验证明,SsDREB能特异性结合DRE顺式作用元件,并能激活下游报告基因的表达;采用农杆菌介导法将SsDREB基因在35S 启动子的驱动下转入烟草中,获得的转基因植株对干旱和盐胁迫抗性均显著提高;为研究SsDREB过量表达提高转基因烟草抗旱、耐盐能力的分子机制,选取烟草中与提高质膜稳定性、消除活性氧和渗透平衡相关的8个逆境胁迫相关基因,以烟草α-tublin基因做为内参基因,利用半定量RT-PCR方法分析在正常生长条件下这8个基因在转基因烟草和对照烟草的表达情况,实验结果表明这8个基因都受外源SsDREB基因的调控,其中6个基因在转基因烟草中的表达量显著高于非转基因植株。

    Abstract:

    SsDREB, which was highly homologous with DREB2 transcription factors from other plants, was cloned from Suaeda salsa L. and its expression was induced by high-salt and drought, not by cold and ABA.In this study, a plant expression vector containing a SsDREB-GFP fusion gene has been constructed and transformed into onion epidermal cells by gene gun method. The result of transient expression showed that the protein encoded by SsDREB was localized in the nucleus. In yeast cells, SsDREB protein was proved to be able to specificly bind to the DRE element and activate the expression of downstream reporter genes. By using agrobacterium-mediated transformation, the SsDREB gene driven by constitutive promoter CaMV 35S was successfully introduced into tobacco (Nicotiana tabacum L.), and the obtained transgenic tabacco plants showed strong drought and salt tolerance. In order to study the molecular mechanism that overexpression of SsDREB increased tolerance to drought and salt stresses in transgenic tobacco, 8 genes from tobacco related with osmotic balance, eliminating active oxygen and improving membrane stability were selected, and their expression in transgenic and control tobacco plants were analyzed by using semi-quantitative RT -PCR method with α -tublin gene as a reference gene under normal growth conditions. The experimental results showed that the 8 genes are regulated by exogenous SsDREB gene, and the expression of 6 genes was significantly higher in transgenic plants than that in non-transgenic plants.

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孙晓波,马鸿翔,刘晓雪,等.盐地碱蓬DREB同源基因SsDREB的功能研究[J].植物遗传资源学报,2015,16(3):624-632.

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  • 收稿日期:2014-05-29
  • 最后修改日期:2014-07-08
  • 录用日期:2015-04-10
  • 在线发布日期: 2015-05-06
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