Biology Group, Discovery, Health & Crop Sciences Research Laboratory, Sumitomo Chemical Co., Ltd., 4-2-1, Takatsukasa, Takarazuka, Hyogo, 665-8555, Japan,
Plant Cell Rep. 2014 Jan;33(1):47-59. doi: 10.1007/s00299-013-1511-5. Epub 2013 Oct 6.
A newly developed copper-inducible gene expression system overcame the mixed results reported earlier, worked well both in cultured cells and a whole plant, and enabled to control flowering timing. Copper is one of the essential microelements and is readily taken up by plants. However, to date, it has rarely been used to control the expression of genes of interest, probably due to the inefficiency of the gene expression systems. In this study, we successfully developed a copper-inducible gene expression system that is based on the regulation of the yeast metallothionein gene. This system can be applied in the field and regulated at approximately one-hundredth of the rate used for registered copper-based fungicides. In the presence of copper, a translational fusion of the ACE1 transcription factor with the VP16 activation domain (VP16AD) of herpes simplex virus strongly activated transcription of the GFP gene in transgenic Arabidopsis. Interestingly, insertion of the To71 sequence, a 5'-untranslated region of the 130k/180k gene of tomato mosaic virus, upstream of the GFP gene reduced the basal expression of GFP in the absence of copper to almost negligible levels, even in soil-grown plants that were supplemented with ordinary liquid nutrients. Exposure of plants to 100 μM copper resulted in an over 1,000-fold induction ratio at the transcriptional level of GFP. This induction was copper-specific and dose-dependent with rapid and reversible responses. Using this expression system, we also succeeded in regulating floral transition by copper treatment. These results indicate that our newly developed copper-inducible system can accelerate gene functional analysis in model plants and can be used to generate novel agronomic traits in crop species.
一个新开发的铜诱导基因表达系统克服了之前报道的混合结果，在培养细胞和整个植物中都能很好地工作，并能控制开花时间。铜是一种必需的微量元素，很容易被植物吸收。然而，到目前为止，它很少被用于控制感兴趣的基因的表达，可能是由于基因表达系统的效率低下。在这项研究中，我们成功地开发了一种基于酵母金属硫蛋白基因调控的铜诱导基因表达系统。该系统可应用于田间，并可在约百分之一的注册铜基杀菌剂使用的速率下进行调节。在铜存在的情况下，ACE1 转录因子与单纯疱疹病毒 VP16 激活结构域（VP16AD）的翻译融合物强烈激活了转基因拟南芥中 GFP 基因的转录。有趣的是，番茄花叶病毒 130k/180k 基因的 5'非翻译区 To71 序列的插入，使 GFP 基因在没有铜的情况下的基础表达降低到几乎可以忽略不计的水平，即使在补充普通液体营养的土壤中生长的植物也是如此。植物暴露于 100μM 铜会导致 GFP 在转录水平上的诱导倍数超过 1000 倍。这种诱导是铜特异性和剂量依赖性的，具有快速和可逆的反应。使用该表达系统，我们还成功地通过铜处理调节了花的转变。这些结果表明，我们新开发的铜诱导系统可以加速模式植物中的基因功能分析，并可用于在作物物种中产生新的农艺性状。
