Selection and Evaluation of Reference Genes for qRT-PCR in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Han, Shipeng; Qin, Qiuju; Da, Wang; Zhou, Yayuan; He, Yunzhuan

doi:10.3390/insects12100902

Cited by 14 publications

(16 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…EF1α and RPL13 are the best reference genes in all the experimental conditions in P. operculella (Lepidoptera: Gelechiidae). Interestingly, in another Lepidoptera insect Spodoptera frugiperda (Noctuidae), based on the online program RefFinder, SOD, RPL10 and RPS24 were reported to be the most stable reference genes for different developmental stages, while α-TUB, RPL10 and ATP were for various tissues, AK, RPL10 and 18S for mating status, 18S and AK for hormone treatment, 18S, RPL10 and SOD for diets treatment, and RPL10, 18S and RPS24 for temperature treatment [67]. The results verified that the expression stability of the reference genes varied under different treatments.…”

Section: Discussionmentioning

confidence: 99%

Reference Genes for Expression Analyses by qRT-PCR in Phthorimaea operculella (Lepidoptera: Gelechiidae)

Shen

Peng

Zhang

et al. 2022

Insects

View full text Add to dashboard Cite

Due to a lack of effective internal references, studies on functional genes in Phthorimaea operculella, a serious Lepidopteran pest attacking potatoes worldwide, have been greatly limited. To select suitable endogenous controls, ten housekeeping genes of actin (ACT), α-tubulin (α-TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), 18S and 28S ribosomal RNA (18S, 28S), ribosomal protein genes RPL4, RPL13 and RPL27 and superoxide dismutase (SOD) were tested. Their expression levels were determined under three different experimental conditions (developmental stages, tissues/organs and temperatures) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). The results clarified that RPL13, EF1α and RPL27 are ranked as the best reference gene combination for measuring gene expression levels among different developing stages and under various temperatures; EF1α and RPL13 are recommended to normalize the gene expression levels among diverse tissues. EF1α and RPL13 are the best reference genes in all the experimental conditions. To validate the utility of the selected reference pair, EF1α and RPL13, we estimated the tissue-biased expression level of chitin synthase A gene (PoChSA). As expected, PoChSA was abundantly expressed in ectodermally derived epidermal cells, and lowly transcribed in the midgut. These findings will lay the foundation for future research on the molecular physiology and biochemistry of P. operculella.

show abstract

Section: Discussionmentioning

confidence: 99%

Reference Genes for Expression Analyses by qRT-PCR in Phthorimaea operculella (Lepidoptera: Gelechiidae)

Shen

Peng

Zhang

et al. 2022

Insects

View full text Add to dashboard Cite

show abstract

“…These traditional used reference genes are not always stably expressed when tested in different species or in a wider range of experimental conditions. For example, the expression stability of the reference gene actin was found to be the least consistent in Spodoptera frugiperda under different treatments, while it was the most stable in Bradysia odoriphaga across varying conditions (Han et al, 2021). In Drosophila melanogaster , high variability in expression of reference genes was observed under different chemical exposures (Matta et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of suitable reference genes for expression analysis using quantitative real‐time polymerase chain reaction in the parasitoid Exorista sorbillans (Diptera: Tachinidae)

Yang

Zhe

et al. 2023

Arch Insect Biochem Physiol

View full text Add to dashboard Cite

The parasitoid Exorista sorbillans (Diptera: Tachinidae) is a larval endoparasitoid of the silkworm Bombyx mori, causing severe damage to silkworm cocoon industry. It is also an important natural enemy resource of insect pests in agriculture and forestry. Despite their roles in biocontrol and pest status on sericulture, there has been limited research on the functional studies of dipteran parasitoids.Quantitative real-time polymerase chain reaction (qRT-PCR) is the most commonly used to address gene functions.Using qRT-PCR, stably expressed reference genes under different experimental conditions are required to normalize the expression of target genes. However, no information regarding suitable qRT-PCR reference genes in dipteran parasitoids has been reported. In this study, we evaluate the expression stability of nine commonly used reference genes in insects including eukaryotic translation elongation factor 1δ (eEF1δ), elongation factor 2, 18S ribosomal RNA (18S rRNA), tubulin 3, actin87, ribosomal protein 49 (RP49), ribosomal protein S15, glyceraldehyde-3-phosphate dehydrogenase, and TATA-box binding protein (TBP) in E. sorbillans under different treatments, including tissues, developmental stages, genders, feeding density and pesticide stress, using ΔC t , BestKeeper, geNorm, Normfinder

show abstract

“…The relative expression levels of genes were normalized by using the 2 −ΔCt method with the reference genes α-TUB and RPL_10 (Table S1). 35 RNA Interference. The small interference RNAs (siRNAs) (sequences listed in Table S1) which are targeting HR3 were designed according to the verified sequences (accession number LOC118279815).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Azadirachtin Inhibits Nuclear Receptor HR3 in the Prothoracic Gland to Block Larval Ecdysis in the Fall Armyworm, Spodoptera frugiperda

Fan,

Wu,

Liu

et al. 2023

J. Agric. Food Chem.

View full text Add to dashboard Cite

Azadirachtin has been used to control agricultural pests for a long time; however, the molecular mechanism of azadirachtin on lepidopterans is still not clear. In this study, the fourth instar larvae of fall armyworm were fed with azadirachtin, and then the ecdysis was blocked in the fourth instar larval stage (L4). The prothoracic glands (PGs) of the treated larvae were dissected for RNA sequencing to determine the effect of azadirachtin on ecdysis inhibition. Interestingly, one of the PG-enriched genes, the nuclear hormone receptor 3 (HR3), was decreased after azadirachtin treatment, which plays a critical role in the 20-hydroxyecdysone action during ecdysis. To deepen the understanding of azadirachtin on ecdysis, the HR3 was knocked out by using the CRISPR/Cas9 system, while the HR3 mutants displayed embryonic lethal phenotype; thus, the stage-specific function of HR3 during larval molting was not enabled to unfold. Hence, the siRNA was injected into the 24 h L4 larvae to knock down HR3. After 96 h, the injected larvae were blocked in the old cuticle during ecdysis which is consistent with the azadirachtin-treated larvae. Taken together, we envisioned that the inhibition of ecdysis in the fall armyworm after the azadirachtin treatment is due to an interference with the expression of HR3 in PG, resulting in larval mortality. The results in this study specified the understanding of azadirachtin on insect ecdysis and the function of HR3 in lepidopteran in vivo.

show abstract

Selection and Evaluation of Reference Genes for qRT-PCR in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Cited by 14 publications

References 42 publications

Reference Genes for Expression Analyses by qRT-PCR in Phthorimaea operculella (Lepidoptera: Gelechiidae)

Reference Genes for Expression Analyses by qRT-PCR in Phthorimaea operculella (Lepidoptera: Gelechiidae)

Evaluation of suitable reference genes for expression analysis using quantitative real‐time polymerase chain reaction in the parasitoid Exorista sorbillans (Diptera: Tachinidae)

Azadirachtin Inhibits Nuclear Receptor HR3 in the Prothoracic Gland to Block Larval Ecdysis in the Fall Armyworm, Spodoptera frugiperda

Contact Info

Product

Resources

About