Developing Portulaca oleracea as a model system for functional genomics analysis of C4/CAM photosynthesis
Renata Callegari Ferrari A , Priscila Pires Bittencourt A , Paula Yumi Nagumo A , Willian Silva Oliveira A , Maria Aurineide Rodrigues A , James Hartwell B and Luciano Freschi
A C
+ Author Affiliations
- Author Affiliations
A Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brasil.
B Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
C Corresponding author. Email: freschi@usp.br
Functional Plant Biology 48(7) 666-682 https://doi.org/10.1071/FP20202
Submitted: 12 July 2020 Accepted: 20 October 2020 Published: 1 December 2020
Abstract
Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C4 and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C4/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C4/CAM under field and laboratory conditions. In the present work, we focussed on developing molecular tools to facilitate functional genomics studies in this species, from the optimisation of RNA isolation protocols to a method for stable genetic transformation. Eleven variations of RNA extraction procedures were tested and compared for RNA quantity and quality. Also, 7 sample sets comprising total RNA from hormonal and abiotic stress treatments, distinct plant organs, leaf developmental stages, and subspecies were used to select, among 12 reference genes, the most stable reference genes for RT-qPCR analysis of each experimental condition. Furthermore, different explant sources, Agrobacterium tumefaciens strains, and regeneration and antibiotic selection media were tested in various combinations to optimise a protocol for stable genetic transformation of P. oleracea. Altogether, we provide essential tools for functional gene analysis in the context of C4/CAM photosynthesis, including an efficient RNA isolation method, preferred reference genes for RT-qPCR normalisation for a range of experimental conditions, and a protocol to produce P. oleracea stable transformants using A. tumefaciens.
Keywords: Agrobacterium tumefaciens, CAM, carbon concentration mechanism, crop biotechnology, Crassulacean acid metabolism, C4 photosynthesis, Portulaca oleracea, purslane, reference gene, stable genetic transformation.
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