A heat-shock inducible system for flexible gene expression in cereals.
A heat-shock inducible system for flexible gene expression in cereals.
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DOI：
10.1186/s13007-020-00677-3
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发表时间：
2020
期刊：
影响因子：
5.1
通讯作者：
Richardson A
中科院分区：
文献类型：
--
作者：
Harrington SA;Backhaus AE;Fox S;Rogers C;Borrill P;Uauy C;Richardson A
使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，基因过度表达的标准方法可能会由于异位基因表达而导致不良的发育表型，甚至胚胎致死。诱导表达系统允许通过阻止这些基因的表达直到用特定诱导剂处理来研究这些基因。当与 Cre-Lox 重组系统结合时，诱导型启动子可用于启动感兴趣基因的组成型表达。然而，虽然这些系统在双子叶植物模型植物（如拟南芥）中得到了很好的应用，但尚未在禾本科植物中得到应用。在这里，我们提出了一种使用 Golden Gate 兼容组件开发的不可逆热休克诱导系统，该系统利用 Cre 重组酶驱动大麦和小麦的组成型基因表达。我们证明 38°C 的热休克处理足以激活构建体并驱动感兴趣基因的表达。调节热休克的持续时间可以控制诱导细胞的密度。短时间的热休克会导致孤立的单细胞中的构建体激活，而较长的持续时间会导致整体构建体激活。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（~ 20°C）下没有表现出激活。该系统为谷类作物的基因功能表征提供了一个适应性强的框架。开发的载体可以轻松适应金门克隆系统中感兴趣的特定基因。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一致和完整应用相关的陷阱。与任何诱导系统一样，必须小心确保预期的构建激活确实发生。
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. 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.
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影响因子：
12.3
作者：
Lawrenson T;Shorinola O;Stacey N;Li C;Østergaard L;Patron N;Uauy C;Harwood W
通讯作者：
Harwood W
影响因子：
7.2
作者：
Joubès, J;De Schutter, K;De Veylder, L
通讯作者：
De Veylder, L
影响因子：
3.5
作者：
Garwick-Coppens SE;Herman A;Harper SQ
通讯作者：
Harper SQ
影响因子：
7.2
作者：
Roslan, HA;Salter, MG;Caddick, MX
通讯作者：
Caddick, MX
DOI：
10.1007/bf00264442
发表时间：
1990-09-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
ODELL, J;CAIMI, P;RUSSELL, S
通讯作者：
RUSSELL, S