Establishment of cell-specifically inducible expression systems in transgenic barley and maize

利用组构或组织特异性启动子过度表达或敲除与植物病理系统相关的基因的实验方法通常会导致多营养效应，如发育异常或生存能力降低。因此，利用这些传统的表达系统在转基因植物中不能很好地研究局限于植物-病原体相互作用背景下的直接基因功能。因此，本项目旨在建立基于细粒曲霉乙醇诱导的AlcRlalcA启动子系统在大麦和玉米中的基因表达体系。这个双组分系统在三个水平上提供了转基因表达的控制：(1)反激活基因AlcR可以由一个适当的细胞特异性启动子驱动，(2)诱导剂可以应用于任何发育阶段，(3)局部局限于所研究植物的一部分。两个候选启动子特异性的高度相关组织在植物与病原体的相互作用将首先通过农杆菌介导转化大麦使用传统的报告基因结构进行功能验证。然后，这些启动子将与反激活子AlcR一起用于开发细胞特异性诱导表达系统。同时，我们将通过农杆菌感染未成熟胚胎或雄激素花粉培养物来建立玉米的遗传转化。最终，设想通过使用alcA启动子驱动GFP基因的细胞特异性诱导表达系统转化大麦和玉米，提供概念证明。在各种后续方法中，所建立的系统将对大麦或玉米与各种致病或互惠真菌的相容相互作用中被认为涉及的基因的功能特征具有很高的价值。

Experimental approaches to over-expression or knock-down of genes implicated in plant pathosystems using constitutive or tissue-specific promoters often result in pleiotrophic effects such as developmental aberrations or reduced viability. Hence, the immediate gene function confined to the context of plant-pathogen interaction cannot be studied properly using these conventional expression systems in transgenic plants. Thus, this project aims at establishing gene expression systems in barley and maize based on the ethanol-inducible AlcRlalcA promoter system derived from Aspergillus nidulans. This two-component system provides control of transgene expression at three levels: (1) the transactivator gene AlcR can be driven by an appropriate cell-specific promoter, (2) the inducing agent can be applied at any developmental stage of choice, and (3) locally confined to only a part of the plant investigated. Two candidate promoters specific for highly relevant tissues in plant-pathogen interactions will be functionally verified first by Agrobacterium-mediated transformation of barley using conventional reporter gene constructs. These promoters will then be used in conjunction with the transactivator AlcR to develop cell-specifically inducible expression systems. In parallel we will establish genetic transformation of maize via infection of either immature embryos or androgenetic pollen cultures with Agrobacteria. Eventually, it is envisaged to provide a proof of concept by transformation of barley and maize using the cell-specifically inducible expression systems with the alcA promoter driving the GFP gene. In various follow-up approaches, the established systems will be highly valuable for functional characterisation of genes putatively implicated in the compatible interaction of barley or maize with various pathogenic or mutualistic fungi.