Evaluation of reference genes for miRNA expression analysis in <i>Galeruca daurica</i> (Coleoptera: Chrysomelidae) using qRT‐PCR

Wang, Haichao; Li, Ling; Li, Yanyan; Tan, Yao; Pang, Bao-Ping

doi:10.1111/1748-5967.12537

Cited by 4 publications

(11 citation statements)

References 39 publications

(44 reference statements)

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“…Our data indicated that let-7-5p shows higher levels of expression in adults than any other developmental stage and lower levels in eggs. Similarly, in G. molesta and G. daurica, a higher expression in adults and lower expression in eggs were observed (Wang X.-W. et al, 2017;Wang et al, 2021).…”

Section: Discussionmentioning

confidence: 91%

“…Based on the data, we found that different programs gave disparate rankings of candidate reference gene stability under identical treatments and distinct rankings of gene stability under different treatments in an identical program. In Galeruca daurica, the best combinations of reference genes were miR-9a-5p and U6 snRNA for different stages of development, miR-305-5p and miR-100-5p for different tissue types, and miR-100-5p and miR-275-3p for various temperature settings (Wang et al, 2021). The best reference gene combinations in Helicoverpa armigera were miR-9 and U6 snRNA for stages of development, miR-305 and miR-100 for tissue types, and miR-100 and miR-92a for temperature treatments, and in Grapholita molesta, the most suitable combinations were miR-16 and U6 snRNA for the stages of development, and miR-281 and U6 snRNA for different settings of temperature (Wang X. et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…For the other tested variables, miR-624 was the most suitable for the stages of development and tissue types and miR-100-5p for sex. In G. daurica, miR-100-5p was the most suitable for the tissue type (Wang et al, 2021). Also, U6 snRNA can be utilized as one suitable reference gene for the quantification of miRNA for different stages of development (Feng et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

“…5SrRNA and U6 snRNA are commonly utilized as reference genes for analyzing the expression of miRNA in insects; however, these genes cannot be consistently appropriate with all treatments (Lim et al, 2011;Xiang et al, 2014). In recent years, screening of reference genes for studies of miRNA in arthropods including Grapholita molesta, Helicoverpa armigera, Bombus lantschouensis, Plutella xylostella, and Galeruca daurica has been performed and found that the reference genes appropriate for assessing the expression of miRNA rely on the species of insect, developmental stage, and environmental conditions, and sometimes, more than one reference gene is needed to normalize the data on gene expression under various abiotic and biotic conditions (Wang X.-W. et al, 2017;Yang et al, 2017;Dong et al, 2019;Zhang et al, 2019;Wang et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

“…In our current work, 11 selected reference genes, including 10 miRNAs (miR-9a, miR-1-3p, miR-11, miR-184, miR-275, miR-100-5p, miR-252a, miR-277, miR-279d, and miR-624) from the B. tabaci databases of small RNA sequencing, have been used for the study of selection and evaluation in previous reports (Wang X.-W. et al, 2017;Yang et al, 2017;Dong et al, 2019;Zhang et al, 2019;Wang et al, 2021). With one commonly utilized reference gene, U6 snRNA, the aforementioned 11 candidates were selected to normalize gene expression data for B. tabaci with insecticide tolerance to 11 popular insecticides used against whiteflies in the field, including pymetrozine, chlorpyrifos, imidacloprid, flupyradifurone, sulfoxaflor, flonicamid, cyantraniliprole, afidopyropen, avermectin, deltamethrin, and β-cypermethrin, and most of them have been recorded as cases of resistance in B. tabaci (Wang et al, 2018a;Wang et al, 2020b,c;Zheng et al, 2021;Wang et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

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Selection and Evaluation of Reference Genes for miRNA Expression Analysis in Bemisia tabaci Under Insecticide Tolerance

Zhang

Gao

et al. 2022

Front. Genet.

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A growing number of studies have focused on the microRNA (miRNA) expression in Bemisia tabaci, one devastating agricultural insect pest of the tropical and subtropical areas for which the primary means of control are insecticides. In studying the genetic underpinnings of insecticide resistance, the choice of stable reference genes for normalizing data plays a key role to acquire unbiased expression profile results from quantitative real-time PCR (qPCR) analysis. Expression profiles of 11 selected reference genes were determined systematically in B. tabaci exposure to 11 insecticides. Furthermore, we assessed the stability of all the selected candidates in relation to other variables including sex, tissue type, and developmental stage. Candidate reference gene validation was conducted by analyzing the let-7-5p expression under various experimental treatments. Five programs BestKeeper, NormFinder, geNorm, △Ct, and RefFinder were applied to verify the accuracy of the selected candidates. Our results displayed that the best choices of the selected candidates for pymetrozine, sulfoxaflor, flonicamid, cyantraniliprole, afidopyropen, and deltamethrin treatment were miR-1-3p and miR-100-5p, U6 and miR-100-5p were best for chlorpyrifos and imidacloprid treatments, and U6 and miR-1-3p were best for flupyradifurone and β-cypermethrin treatments. The reference genes miR-624, miR-252, and miR-275 worked best in adult tissues, miR-100-5p and miR-1-3p worked best in either sex, and miR-624 and miR-11 were best to use across developmental stages. Not even one reference gene was found to be suitable for all experimental conditions. Our results contributed to the growing body of the literature on qPCR reference gene selection under various experimental conditions and facilitate further investigation on gene expression changes in B. tabaci, resulting from pesticide exposure.

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Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Selection and Evaluation of Reference Genes for miRNA Expression Analysis in Bemisia tabaci Under Insecticide Tolerance

Zhang

Gao

et al. 2022

Front. Genet.

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MicroRNA miR‐285 modulates the metamorphosis in Galeruca daurica by targeting Br‐C

Wang,

Li,

Zhang

et al. 2024

Pest Management Science

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BACKGROUDGaleruca daurica has become a new pest on the Inner Mongolia grasslands since its abrupt outbreak in 2009, and caused serious damage. As a pupa indicator during insect metamorphosis, the early response gene of ecdysone signaling pathway, Broad‐Complex (Br‐C), plays a vital role in the growth and development of insects. MicroRNAs (miRNA) are small non‐coding RNAs which mediate various biological activities. However, it is unknown whether and how Br‐C is regulated by miRNAs.RESULTSTemporal expression profiles revealed that miR‐285 and Br‐C basically displayed an opposite trend during the larval‐adult development, and Br‐C was sharply up‐regulated on the last day of final instar larvae meanwhile miR‐285 was significantly down‐regulated. Both dual‐luciferase reporter assay and miRNA‐mRNA interaction assay indicated that miR‐285 interacts with the coding sequence of Br‐C and represses its expression. Not only overexpression but also downexpression of miR‐285 led to the failure of larval to pupal to adult metamorphosis. In addition, both overexpression of miR‐285 and silence of Br‐C inhibited the expression of Br‐C and other ecdysone signaling pathway genes including E74, E75, ECR, FTZ‐F1, and HR3. On the contrary, suppressing miR‐285 obtained opposite results. Further experiments showed that 20‐hydroxyecdysone down‐regulated miR‐285, and up‐regulated Br‐C and above‐mentioned genes whereas JHA resulted in opposite effects.CONCLUSIONOur results reveal that miR‐285 is involved in mediating the metamorphosis in G. daurica by targeting Br‐C in the ecdysone signaling pathway. miR‐285 and its target Br‐C could be as a potential target for G. daurica management.

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Comparative Transcriptome Analysis of Galeruca daurica Reveals Cold Tolerance Mechanisms

Zhang,

Sun,

Zhang

et al. 2023

Genes

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Galeruca daurica (Joannis) is a pest species with serious outbreaks in the Inner Mongolian grasslands in recent years, and its larvae and eggs are extremely cold-tolerant. To gain a deeper understanding of the molecular mechanism of its cold-tolerant stress response, we performed de novo transcriptome assembly of G. daurica via RNA-Seq and compared the differentially expressed genes (DEGs) of first- and second-instar larvae grown and developed indoors and outdoors, respectively. The results show that cold tolerance in G. daurica is associated with changes in gene expression mainly involved in the glycolysis/gluconeogenesis pathway, the fatty acid biosynthesis pathway and the production of heat shock proteins (HSPs). Compared with the control group (indoor), the genes associated with gluconeogenesis, fatty acid biosynthesis and HSP production were up-regulated in the larvae grown and developed outdoors. While the changes in these genes were related to the physiological metabolism and growth of insects, it was hypothesized that the proteins encoded by these genes play an important role in cold tolerance in insects. In addition, we also investigated the expression of genes related to the metabolic pathway of HSPs, and the results show that the HSP-related genes were significantly up-regulated in the larvae of G. daurica grown and developed outdoors compared with the indoor control group. Finally, we chose to induce significant expression differences in the Hsp70 gene (Hsp70A1, Hsp70-2 and Hsp70-3) via RNAi to further illustrate the role of heat stress proteins in cold tolerance on G. daurica larvae. The results show that separate and mixed injections of dsHSP70A1, dsHsp70-2 and dsHsp70-3 significantly reduced expression levels of the target genes in G. daurica larvae. The super-cooling point (SCP) and the body fluid freezing point (FP) of the test larvae were determined after RNAi using the thermocouple method, and it was found that silencing the Hsp70 genes significantly increased the SCP and FP of G. daurica larvae, which validated the role of heat shock proteins in the cold resistance of G. daurica larvae. Our findings provide an important theoretical basis for further excavating the key genes and proteins in response to extremely cold environments and analyzing the molecular mechanism of cold adaptation in insects in harsh environments.

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Evaluation of reference genes for miRNA expression analysis in Galeruca daurica (Coleoptera: Chrysomelidae) using qRT‐PCR

Cited by 4 publications

References 39 publications

Selection and Evaluation of Reference Genes for miRNA Expression Analysis in Bemisia tabaci Under Insecticide Tolerance

Selection and Evaluation of Reference Genes for miRNA Expression Analysis in Bemisia tabaci Under Insecticide Tolerance

MicroRNA miR‐285 modulates the metamorphosis in Galeruca daurica by targeting Br‐C

Comparative Transcriptome Analysis of Galeruca daurica Reveals Cold Tolerance Mechanisms

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