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

Author(s)

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

• Document type: Journal article
• Source: Plant Physiology and Biochemistry; v. 171, p. 12-pg., 2022-01-07.

Abstract

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)

• FAPESP's process: 18/08215-0 - Study of post-transcriptional gene regulation processes in the somatic embryogenesis in Araucaria angustifolia
• Grantee: Leandro Francisco de Oliveira
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/26961-2 - Secondary metabolism during ontogeny of Piper species and their implication in herbivory
• Grantee: Dimitre Alexandrov Ivanov
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 20/03791-2 - Dimensions US-Biota São Paulo: chemically mediated multi-trophic interaction diversity across tropical gradients
• Grantee: Amanda Rusiska Piovezani
• Support Opportunities: Scholarships in Brazil - Technical Training Program - Technical Training