A heat-shock inducible system for flexible gene expression in cereals
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Plant Methods Open Access
METHODOLOGY
A heat‑shock inducible system for flexible gene expression in cereals Sophie A. Harrington1, Anna E. Backhaus1, Samantha Fox1, Christian Rogers2, Philippa Borrill3, Cristobal Uauy1* and Annis Richardson1,4*
Abstract Background: 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 Arabidopsis thaliana, they have not yet been implemented in grasses. Results: 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). Conclusions: 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. Keywords: Inducible promoters, Cre recombinase, Transgenic, Inducible expression, Clonal sectors, Wheat, Barley, Grasses Background Historically, transformation in cereal crops such as wheat and barley has been hampered by low efficiencies and high costs [1]. Yet recent advances in transformation techniques have shown remarkable improvements of transformation efficiency [1, 2]. As a result, functional characterisation of cereal genes increasingly uses *Correspondence: [email protected]; [email protected] 1 John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK Full list of author information is available at the end of the article
transgenic approaches. Commonly, genes are characterised using standard over-expression systems, where the gene of interest is placed under the control of a constitutive promoter [3]. In some cases, how
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