Developing Portulaca oleracea as a model system for functional genomics analysis of C-4/CAM photosynthesis

Autor(es)

Ferrari, Renata Callegari ^[1] ; Bittencourt, Priscila Pires ^[1] ; Nagumo, Paula Yumi ^[1] ; Oliveira, Willian Silva ^[1] ; Rodrigues, Maria Aurineide ^[1] ; Hartwell, James ^[2] ; Freschi, Luciano ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Biociencias, Dept Bot, BR-05508090 Sao Paulo - Brazil

^[2] Univ Liverpool, Inst Syst Mol & Integrat Biol, Dept Biochem & Syst Biol, Liverpool L69 7ZB, Merseyside - England

• Tipo de documento: Artigo Científico
• Fonte: FUNCTIONAL PLANT BIOLOGY; v. 48, n. 7 DEC 2020.
• Citações Web of Science: 1

Resumo

Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C-4 and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C-4/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C-4/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 C-4/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. (AU)

• Processo FAPESP: 16/04755-4 - Investigando a compatibilidade entre as fotossínteses C4 e CAM em um mesmo indivíduo: mudanças no perfil transcricional global, regulação hormonal e localização tecidual da maquinaria C4 e CAM em Portulaca oleracea
• Beneficiário: Renata Callegari Ferrari
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto