Dataset Information

Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae).

ABSTRACT: Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the sensitive method to quantify the expression levels of target genes on the basis of endogenous control. An appropriate reference gene set for normalization is essential for reliable results. The ghost moth, Thitarodes armoricanus, a host species of a medicinal fungus, Ophiocordyceps sinensis, is an economically important member of the Lepidoptera. Recent studies have focused on the mechanism of adaptation of this species to its high-altitude environment and host immune response to O. sinensis infection and RT-qPCR is commonly used in these studies to decipher the genetic basis of physiological functions. However, a thorough assessment of candidate reference genes in the genus Thitarodes is lacking. Here, the expression levels of eight candidate reference genes (ACT, EF, EIF4A, GAPDH, G6PDH, RPL13A, TUB and 18S) in T. armoricanus at different developmental stages and in different body parts of the seventh instar larvae were analyzed, along with larvae kept under low temperatures, larvae exposed to two fungal infections and larvae fed different diets. Three established software programs-Bestkeeper, geNorm and NormFinder-were employed to calculate variation among the treatments. The results revealed that the best-suited reference genes differed across the treatments, with EF, EIF4A and GAPDH found to be the best suited for the different developmental stages and larvae body parts; EF, EIF4A and RPL13A found to be the best suited for low-temperature challenge; and EF, EIF4A and TUB found to be the best suited for the fungal infections and dietary treatments. This study thus further contributes to the establishment of an accurate method for normalizing RT-qPCR results for T. armoricanus and serves as a reference for gene expression studies of related insect species.

SUBMITTER: Liu G

PROVIDER: S-EPMC4938418 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae).

Liu Guiqing G Qiu Xuehong X Cao Li L Zhang Yi Y Zhan Zubing Z Han Richou R

PloS one 20160708 7

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the sensitive method to quantify the expression levels of target genes on the basis of endogenous control. An appropriate reference gene set for normalization is essential for reliable results. The ghost moth, Thitarodes armoricanus, a host species of a medicinal fungus, Ophiocordyceps sinensis, is an economically important member of the Lepidoptera. Recent studies have focused on the mechanism of adaptation of t ...[more]

PMID: 27392023

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Project description:BackgroundLepidoptera encompasses more than 160,000 described species that have been classified into 45-48 superfamilies. The previously determined Lepidoptera mitochondrial genomes (mitogenomes) are limited to six superfamilies of the lineage Ditrysia. Compared with the ancestral insect gene order, these mitogenomes all contain a tRNA rearrangement. To gain new insights into Lepidoptera mitogenome evolution, we sequenced the mitogenomes of two ghost moths that belong to the non-ditrysian lineage Hepialoidea and conducted a comparative mitogenomic analysis across Lepidoptera.ResultsThe mitogenomes of Thitarodes renzhiensis and T. yunnanensis are 16,173 bp and 15,816 bp long with an A + T content of 81.28 % and 82.34 %, respectively. Both mitogenomes include 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and the A + T-rich region. Different tandem repeats in the A + T-rich region mainly account for the size difference between the two mitogenomes. All the protein-coding genes start with typical mitochondrial initiation codons, except for cox1 (CGA) and nad1 (TTG) in both mitogenomes. The anticodon of trnS(AGN) in T. renzhiensis and T. yunnanensis is UCU instead of the mostly used GCU in other sequenced Lepidoptera mitogenomes. The 1,584-bp sequence from rrnS to nad2 was also determined for an unspecified ghost moth (Thitarodes sp.), which has no repetitive sequence in the A + T-rich region. All three Thitarodes species possess the ancestral gene order with trnI-trnQ-trnM located between the A + T-rich region and nad2, which is different from the gene order trnM-trnI-trnQ in all previously sequenced Lepidoptera species. The formerly identified conserved elements of Lepidoptera mitogenomes (i.e. the motif 'ATAGA' and poly-T stretch in the A + T-rich region and the long intergenic spacer upstream of nad2) are absent in the Thitarodes mitogenomes.ConclusionThe mitogenomes of T. renzhiensis and T. yunnanensis exhibit unusual features compared with the previously determined Lepidoptera mitogenomes. Their ancestral gene order indicates that the tRNA rearrangement event(s) likely occurred after Hepialoidea diverged from other lepidopteran lineages. Characterization of the two ghost moth mitogenomes has enriched our knowledge of Lepidoptera mitogenomes and contributed to our understanding of the mechanisms underlying mitogenome evolution, especially gene rearrangements.

Dataset Information

Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae).

Publications

Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae).

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