Harrington Sophie A, Backhaus Anna E, Fox Samantha, Rogers Christian, Borrill Philippa, Uauy Cristobal, Richardson Annis
John Innes Centre, Norwich Research Park, Norwich, NR4 7UH UK.
ENSA, Sainsbury Laboratory, University of Cambridge, Cambridge, CB2 1LR UK.
Plant Methods. 2020 Oct 14;16:137. doi: 10.1186/s13007-020-00677-3. eCollection 2020.
Functional characterisation of genes using transgenic methods is increasingly common in cereal crops. Yet standard methods of gene over-expression can lead to undesirable developmental phenotypes, or even embryo lethality, due to ectopic gene expression. Inducible expression systems allow the study of such genes by preventing their expression until treatment with the specific inducer. When combined with the Cre-Lox recombination system, inducible promoters can be used to initiate constitutive expression of a gene of interest. Yet while these systems are well established in dicot model plants, like , they have not yet been implemented in grasses.
Here we present an irreversible heat-shock inducible system developed using Golden Gate-compatible components which utilises Cre recombinase to drive constitutive gene expression in barley and wheat. We show that a heat shock treatment of 38 °C is sufficient to activate the construct and drive expression of the gene of interest. Modulating the duration of heat shock controls the density of induced cells. Short durations of heat shock cause activation of the construct in isolated single cells, while longer durations lead to global construct activation. The system can be successfully activated in multiple tissues and at multiple developmental stages and shows no activation at standard growth temperatures (~ 20 °C).
This system provides an adaptable framework for use in gene functional characterisation in cereal crops. The developed vectors can be easily adapted for specific genes of interest within the Golden Gate cloning system. By using an environmental signal to induce activation of the construct, the system avoids pitfalls associated with consistent and complete application of chemical inducers. As with any inducible system, care must be taken to ensure that the expected construct activation has indeed taken place.
利用转基因方法对基因进行功能表征在谷类作物中越来越普遍。然而，由于异位基因表达，标准的基因过表达方法可能导致不良的发育表型，甚至胚胎致死。诱导表达系统通过在使用特定诱导剂处理之前阻止基因表达，从而允许对这类基因进行研究。当与Cre-Lox重组系统结合使用时，诱导型启动子可用于启动目标基因的组成型表达。然而，虽然这些系统在双子叶模式植物（如 ）中已得到充分确立，但尚未在禾本科植物中得到应用。
在此，我们展示了一种使用与金门兼容的组件开发的不可逆热激诱导系统，该系统利用Cre重组酶在大麦和小麦中驱动目标基因的组成型表达。我们表明，38°C的热激处理足以激活构建体并驱动目标基因的表达。调节热激持续时间可控制诱导细胞的密度。短时间的热激会导致分离的单个细胞中构建体的激活，而较长时间则会导致整体构建体的激活。该系统可以在多个组织和多个发育阶段成功激活，并且在标准生长温度（约20°C）下无激活现象。
该系统为谷类作物基因功能表征提供了一个适应性框架。所开发的载体可以很容易地在金门克隆系统中针对特定的目标基因进行改造。通过使用环境信号来诱导构建体的激活，该系统避免了与化学诱导剂的持续和完全应用相关的问题。与任何诱导系统一样，必须注意确保确实发生了预期的构建体激活。
