Evaluation and Validation of Reference Genes for qRT-PCR Normalization in Frankliniella occidentalis (Thysanoptera:Thripidae)

2019

Entomological Science

Selecting stable reference genes for reverse transcription quantitative real‐time polymerase chain reaction analysis is critically important for gene expression. Bradysia odoriphaga is the most serious pest of Chinese chives, but our knowledge of its genetics is limited. In the present study, five housekeeping genes (β‐Actin, α‐Tubulin, RPS7, GAPDH and 18S rRNA) were cloned from B. odoriphaga using the combined techniques of reverse transcription polymerase chain reaction with rapid amplification of cDNA ends. The five genes, together with RPS15, were quantified for transcription stability in B. odoriphaga under various experimental conditions. Their expression stability was evaluated using four different algorithms (a comparative ΔCt method, GeNorm, NormFinder and BestKeeper). Additionally, we used an online Web‐based tool (RefFinder) to assign an overall final rank to each candidate gene. These analyses identified 18S, RPS15 and RPS7 as the most stable reference genes across developmental stages. 18S, RPS7 and Tubulin were the most stable reference genes for monitoring gene expression across different tissues of adults. RPS7, GAPDH and Tubulin were selected as the best reference genes across different larval tissues. GAPDH and RPS15 were selected to normalize gene expression for experiments of insecticide treatments. Actin and GAPDH are considered suitable reference genes in experiments of temperature treatments. Actin and Tubulin are considered suitable reference genes for starvation experiments. This study provides an important supplement of suitable reference genes for B. odoriphaga and these results provide clues toward the understanding of the genes’ biological functions in B. odoriphaga.

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Selection of reference genes for quantitative real‐time polymerase chain reaction normalization in Bradysia odoriphaga (Diptera: Sciaridae)

Tang

Dai

2019

Entomological Science

“…The mRNA level of the two hsc70s was measured by comparative quantitative real-time PCR amplification. The 18S rRNA and GAPDH were used as internal standard for cold and heat treatments respectively (Zheng et al 2014). PCR reactions were performed in 20-μL reaction volumes that included iTaq Universal SYBR Green Supermix (2×) (Bio-Rad, USA), 1 μL of each forward and reverse primer (10 μmol L -1 ) (Table 1), 2 μL of cDNA template (2.5×10 -4 μg μL -1 ), and 6 μL…”

Section: Quantitative Real-time Reverse Transcriptase Pcr (Qrt-pcr) Amentioning

confidence: 99%

“…in AF and AM at different heat and cold temperatures, the relative mRNA levels of the two genes were quantified by using real-time quantitative PCR. GAPDH and 18S were used as candidate reference genes in heat and cold stress, respectively (Zheng et al 2014).…”

Section: Genomic Structure Analysismentioning

confidence: 99%

Characterization of two novel heat shock protein 70s and their transcriptional expression patterns in response to thermal stress in adult of Frankliniella occidentalis (Thysanoptera: Thripidae)

Qin

Gao

Journal of Integrative Agriculture

et al. 2018

Self Cite

Heat shock protein 70 (HSP70) is one of the most important members in the heat shock protein family, and plays important roles in the thermotolerance of insect. To explore the molecular mechanism of thermotolerance of Frankliniella occidentalis adults, the difference in the expression of HSP70s in F. occidentalis male or female adults under the thermal stress was studied under the laboratory conditions. Two full length cDNAs of HSP70s gene (Fohsc704 and Fohsc705) were cloned from F. occidentalis by using RT-PCR and RACE. The genomic sequence was demonstrated by genomic validation, and the position and size of the intron were analyzed by sequence analysis of cDNA. Real-time PCR was used to analyze the HSP70 expression patterns. The cDNA of Fohsc704 and Fohsc705 possessed 2 073 and 1 476 bp which encoded 690 and 491 amino acids (aa) with a calculated molecular weight of 75 and 54 kDa, respectively. Four introns in Fohsc704 and six introns in Fohsc705 protein were found. However, the HSP70 protein sequences in our study were ended with EKKN and GIFL, which were different from the reported FoHSP70s. Various expression patterns of Fohsc704 and Fohsc705 were found in both genders of F. occidentalis under thermal stress. The expression of Fohsc704 and Fohsc705 reached to the highest level at-12 and-8°C in male adults, respectively, and Fohsc705 expressed the highest level at 33°C in female adults. In conclusion, HSP70s of F. occidentalis in our study are novel heat shock proteins. There were difference in expression patterns of the two hsc70s in genders of F. occidentalis, and the two HSP70s play important roles in the thermotolerance of F. occidentalis.

“…E, egg; N1, sex undifferentiated 1st-instar nymphs; N2, sex undifferentiated 2ndinstar nymphs; N2♂ and N2♀, 2nd-instar of male and female nymphs, respectively; N3♀, 3rd-instar of female nymph; Pre, male prepupa; P, male pupa; A♂ and A♀, male and female adults, respectively. five softwares have been widely used to identify reference genes in different insects, including the commonly used model insect Drosophila melanogaster (Ponton et al 2011), medical insect Cimex lectularius (Mamidala et al 2011), economic insect Bombus terrestris (Niu et al 2014), crop pest Nilaparvata lugens (Yuan et al 2014;Wan et al 2017), and vegetable pests (Zheng et al 2014). These studies have substantiated the importance of reference gene screening and validation to be included when conducting gene expression analyses.…”

Section: Introductionmentioning

confidence: 99%

Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development

Zheng

Journal of Integrative Agriculture

et al. 2019

Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ∆Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18S were selected as the most stable genes by both the ∆Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase (JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.