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

参考基因 生物 基因 基因表达 遗传学 转录组 亲环素 基因表达谱 肽基脯氨酰异构酶 候选基因 计算生物学 生物化学 分子生物学 异构酶
作者
Leandro Francisco de Oliveira,Amanda Rusiska Piovezani,Dimitre A. Ivanov,Leonardo Yoshida,Eny Iochevet Segal Floh,Massuo Jorge Kato
出处
期刊:Plant Physiology and Biochemistry [Elsevier]
卷期号:171: 201-212 被引量:9
标识
DOI:10.1016/j.plaphy.2021.12.033
摘要

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.
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