Selection and validation of reference genes for measuring gene expression in Piper species at different life stages using RT-qPCR analysis

Autor(es)

de Oliveira, Leandro Francisco ; Piovezani, Amanda Rusiska ; Ivanov, Dimitre A. ; Yoshida, Leonardo ; Segal Floh, Eny Iochevet ; Kato, Massuo Jorge

• Tipo de documento: Artigo Científico
• Fonte: Plant Physiology and Biochemistry; v. 171, p. 12-pg., 2022-01-07.

Resumo

The secondary metabolism of Piper species is known to produce a myriad of natural products from various biosynthetic pathways which, represent a rich source of previously uncharacterized chemical compounds. The determination of gene expression profiles in multiple tissue/organ samples could provide valuable clues towards understanding the potential biological functions of chemical changes in these plants. Studies on gene expression by RT-qPCR require particularly careful selection of suitable reference genes as a control for normalization. Here, we provide a study for the identification of reliable reference genes in P. arboreum, P. gaudichaudianum, P. malacophyllum, and P. tuberculatum, at two different life stages: 2-month-old seedlings and adult plants. To do this, annotated sequences were recovered from transcriptome datasets of the above listed Piper spp. These sequences were subjected to expression analysis using RT-qPCR, followed by analysis using the geNorm and NormFinder algorithms. A set of five genes were identified showing stable expression: ACT7 (Actin-7), Cyclophilin (Peptidyl-prolyl cis-trans isomerase), EF1 alpha (Elongation factor 1-alpha), RNABP (RNA-binding protein), and UBCE (Ubiquitin conjugating enzyme). The universality of these genes was then validated using two target genes, ADC (arginine decarboxylase) and SAMDC (S-adenosylmethionine decarboxylase), which are involved in the biosynthesis of polyamines. We showed that normalization genes varied according to Piper spp., and we provide a list of recommended pairs of the best combination for each species. This study provides the first set of suitable candidate genes for gene expression studies in the four Piper spp. assayed, and the findings will facilitate subsequent transcriptomic and functional gene research. (AU)

• Processo FAPESP: 18/08215-0 - Estudo dos processos de regulação gênica pós-transcricional na embriogênese somática em Araucaria angustifolia
• Beneficiário: Leandro Francisco de Oliveira
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 15/26961-2 - Secondary metabolism during ontogeny of Piper species and their implication in herbivory
• Beneficiário: Dimitre Alexandrov Ivanov
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 20/03791-2 - Desenvolvimento e integração de ferramentas computacionais para a análise de transcriptoma de espécies de Piper
• Beneficiário: Amanda Rusiska Piovezani
• Modalidade de apoio: Bolsas no Brasil - Programa Capacitação - Treinamento Técnico