Baros Christopher J, Beerkens Jeremy, Ludwig Martha
School of Molecular Sciences, University of Western Australia, Crawley, WA, 6009, Australia.
Plant Methods. 2024 Dec 27;20(1):193. doi: 10.1186/s13007-024-01306-z.
The genus Flaveria has been studied extensively as a model for the evolution of C photosynthesis. Thus far, molecular analyses in this genus have been limited due to a dearth of genomic information and the lack of a rapid and efficient transformation protocol. Since their development, Agrobacterium-mediated transient transformation protocols have been instrumental in understanding many biological processes in a range of plant species. However, this technique has not been applied to the genus Flaveria. Here, an efficient protocol for the Agrobacterium-mediated transient transformation of the leaves of the C species Flaveria bidentis is presented. This technique has the distinct advantages of rapid turnaround, the ability to co-transform with multiple constructs, and the capacity to assay coding and non-coding regions of Flaveria genomes in a homologous context. To illustrate the utility of this protocol, the quantitative transcriptional regulation of phosphoenolpyruvate carboxylase, the primary carboxylase of C plants, was investigated. A 24 bp region in the ppcA1 proximal promoter was found to elicit high levels of reporter gene expression. The Agrobacterium-mediated transient transformation of F. bidentis leaves will accelerate the understanding of the biology and evolution of C photosynthesis in the genus Flaveria as well as in other C lineages.
黄顶菊属已被广泛研究，作为C4光合作用进化的模型。到目前为止，由于缺乏基因组信息以及缺乏快速有效的转化方案，该属的分子分析受到限制。自农杆菌介导的瞬时转化方案开发以来，它们在理解一系列植物物种的许多生物学过程中发挥了重要作用。然而，这项技术尚未应用于黄顶菊属。在此，我们提出了一种高效的农杆菌介导的C4物种二齿黄顶菊叶片瞬时转化方案。该技术具有周转快、能够与多个构建体共转化以及能够在同源背景下分析黄顶菊属基因组的编码和非编码区域的显著优点。为了说明该方案的实用性，我们研究了C4植物的主要羧化酶磷酸烯醇式丙酮酸羧化酶的定量转录调控。在ppcA1近端启动子中发现一个24bp的区域可引发高水平的报告基因表达。农杆菌介导的二齿黄顶菊叶片瞬时转化将加速对黄顶菊属以及其他C4谱系中C4光合作用的生物学和进化的理解。
