Reference genes selection for quantitative gene expression studies in tea green leafhoppers, Empoasca onukii Matsuda

Abstract

Empoasca onukii Matsuda is one of the most devastating pests of the tea plant (Camellia sinensis). Still, the presumed expression stability of its reference genes (RGs) has not been analyzed. RGs are essential for accurate and reliable gene expression analysis, so this absence has hampered the study of the insect’s molecular biology. To find candidate RGs for normalizing gene expression data, we cloned ten common housekeeping genes from E. onukii. Using the ΔCt method, geNorm, NormFinder and BestKeeper, we screened the RGs that were appropriate for quantifying the mRNA transcription of cellular responses under five experimental conditions. We identified the combinations of α-TUB and G6PDH, α-TUB and UBC, two RGs (α-TUB and β-TUB1) or three RGs (α-TUB, RPL13 and GAPDH), AK and UBC, or RPL13 and α-TUB as the best for analyzing gene expression in E. onukii adults of both sexes in different tissues, nymphs at different developmental stages, nymphs exposed to different temperatures or nymphs exposed to photoperiod stress. Finally, the E. onukii cysteine proteinase (Eocyp) was chosen as the target gene to validate the rationality of the proposed RGs. In conclusion, our study suggests a series of RGs with which to study the gene expression profiles of E. onukii that have been manipulated (biotically or abiotically) using reverse transcription quantitative polymerase chain reaction. The results offer a solid foundation for further studies of the molecular biology of E. onukii.

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

“…α-TUB is the main component of microtubules that form the cytoskeleton structure, which plays an important role in several eukaryotic cellular processes, including cell mobility and division, and intracellular trafficking 29 . Our results are consistent with previous studies reporting that α-TUB was stable for developmental stages, sexes, tissues, temperature and photoperiod stresses in Empoasca onukii 8 , among various tissues from Coleomegilla maculata 30 and Mythimna separata 24 , for different temperatures in Phenacoccus solenopsis 31 as well as for developmental stages, tissues and sexes in Hermetia illucens 32 . The RPL12 gene encodes a structural protein of ribosomes and is involved in protein translation 33 .…”

“…Although β-ACT and ArgK are traditional reference genes and have been used in many gene expression studies, they are not always stably expressed. Consistent with our results, many studies have shown unstable expression of β-ACT under variable conditions in a variety of species, including Bemisia tabaci 40 , M. separata 35 , F. occidentalis 39 , Henosepilachna vigintioctopunctata 41 , P. japonica 38 , Hippodamia convergens 42 and C. maculata 30 , as well as for ArgK in Spodoptera litura 43 , P. japonica 38 , C. maculata 30 and E. onukii 8 .…”

“…Based on a literature search, we selected nine genes (β-ACT, ArgK, EF1-α, GAPDH, RPL12, RPS23, α-TUB, 18S and 28S) commonly used as reference genes and that have shown high stability in other insect species to investigate their suitability as reference genes for RT-qPCR in A. eugenii 7,8,30,34,43,46 . Degenerate primers for RPS23, 18S, 28S and RpII140 were manually designed based on conserved nucleotide sequence among other Coleoptera species.…”

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plants under differential biotic stresses

“…Multiple RGs have been suggested for normalizing target gene expression, which will reduce the probability of biased normalization 13,48 . In the current study, our results demonstrated using multiple RGs simultaneously in qRT-PCR analysis would increase the sensitivity of gene expression in E. onukii infested leaves (Fig.…”

Reference gene selection for quantitative gene expression analysis in black soldier fly (Hermetia illucens)

“…The selected environmental conditions ranged from biotic to abiotic treatments. Biotic conditions included developmental stage, tissue and sex, which were commonly used in the study of reference genes stability [23, 26, 27]. Abiotic conditions included food, heavy metals and antibiotics.…”