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

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

doi:10.1016/j.plaphy.2021.12.033

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

Plant Physiol Biochem. 2022 Jan 15:171:201-212. doi: 10.1016/j.plaphy.2021.12.033. Epub 2021 Dec 30.

Authors

Leandro Francisco de Oliveira¹, Amanda Rusiska Piovezani², Dimitre A Ivanov³, Leonardo Yoshida⁴, Eny Iochevet Segal Floh⁵, Massuo Jorge Kato⁴

Affiliations

¹ Laboratory of Plant Cell Biology, Department of Botany, Institute of Biosciences, University of São Paulo, Rua Do Matão, 277, 05508-090, São Paulo, SP, Brazil.
² Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo, 05508-900, São Paulo, Brazil; Laboratory of Plant Cell Biology, Department of Botany, Institute of Biosciences, University of São Paulo, Rua Do Matão, 277, 05508-090, São Paulo, SP, Brazil.
³ Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo, 05508-900, São Paulo, Brazil; Department of Biology, University of Western Ontario, 1151 Richmond St, London, ON, Canada, N6A 3K7.
⁴ Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo, 05508-900, São Paulo, Brazil.
⁵ Laboratory of Plant Cell Biology, Department of Botany, Institute of Biosciences, University of São Paulo, Rua Do Matão, 277, 05508-090, São Paulo, SP, Brazil. Electronic address: enyfloh@usp.br.

PMID: 35007951
DOI: 10.1016/j.plaphy.2021.12.033

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), EF1α (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.

Keywords: Gene expression; Housekeeping genes; Piper; RT-qPCR; Reference gene; Validation; qPCR.

MeSH terms

Gene Expression Profiling
Gene Expression Regulation, Plant
Piper* / genetics
Real-Time Polymerase Chain Reaction
Reference Standards
Transcriptome