DOI: 10.5061/dryad.866t1g1sv
title: Characterization of hypoxia-inducible factor α and its response to hypoxia in Callosobruchus chinensis (Coleoptera, Bruchidae)

description: Hypoxia-inducible factor (HIF) plays an essential role in hypoxic
 adaptation in various organisms. Callosobruchus chinensis, a common food
 storage pest, is highly adapted to hypoxic conditions, while the
 underlying mechanism of which is currently unclear. Here, we isolated,
 sequenced, and bioinformatically analyzed HIF1α from C. chinensis, then
 examined its mRNA and protein expression profiles under hypoxic conditions
 for various developmental stages and tissue types. The effects of
 environmental hypoxia on other genes in the HIF pathway response,
 aspartyl-hydroxylase (FIH) and prolyl hydroxylase (PHD), were also
 examined. We found that FIH and PHD were also highly conserved and that
 their expression varied with developmental stage, tissue type, and
 environmental hypoxia. Overall, hypoxia caused an increase in HIF1α and
 FIH mRNA expression, and a decrease in PHD mRNA expression, while
 expression of their proteins showed different in response to hypoxia.
 HIF1α protein levels increased, PHD and FIH protein levels decreased under
 hypoxic conditions, compared to normoxic conditions. This study has
 preliminarily investigated the bioinformation of C. chinensis HIF1α
 response relationship between HIF1α and its hydroxylases under normoxic
 and hypoxic conditions, which might provide important clues for future
 work on clarifying the mechanism of stored-product insect unique hypoxia
 adaption.
description: <strong>Quantitative real-time
 PCR (qRT-PCR)</strong> qRT-PCR was used to
 examine the expression levels of genes related to the HIF pathway in
 various tissues and developmental stages, as described previously.
 Briefly, 1 μg of total RNA was reverse-transcribed to cDNA using the
 PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio). Three genes
 were amplified using SYBR Premix Ex Taq™ II Kit (Takara Bio) in a CFX
 connect Real-Time system (Bio-Rad, Hercules, CA, USA). β-actin was
 identified via RNA-Seq analysis and used as a reference gene. Primer
 specificity was examined by dissociation curve analysis; fold-change
 values were calculated using the 2<sup>−ΔΔCt</sup> method
 (Schmittgen and Livak, 2008). Each experiment was performed in triplicate
 with two technical replicates. The primers used for qRT-PCR are presented
 in Table S4. <strong>Western blot
 analysis</strong><em> </em>
 Approximately 100 mg
 <em>C. chinensis</em> larvae or tissues were suspended in 1 mL
 of RIPA lysis buffer in 2 mL microtubes. The lysed tissues were then
 homogenized using a Tissue Lyser (Qiagen, Hilden, Germany) and centrifuged
 at 4°C, 12,000 rpm for 5 min; the protein-containing supernatant was used
 for downstream applications. The protein content of the supernatant was
 quantified using Bradford reagent (Bio-Rad). Then, 20 μg of protein
 suspended in loading buffer was analyzed by sodium dodecyl
 sulfate–polyacrylamide gel electrophoresis on 5% gels, followed by Western
 blotting onto a polyvinylidene difluoride (PVDF) membrane. The PVDF
 membrane containing the protein samples was washed three times with
 tris-buffered saline Tween-20 (TBST) buffer, and then incubated at 25°C
 for 2 h with the primary antibody anti-HIF1α (Boster, Pleasanton, CA, USA)
 at 1:500 dilution, with anti-glyceraldehyde 3-phosphate dehydrogenase
 (Boster) at 1:2,000 dilution used as a loading control. The membrane was
 washed three times with TBST buffer, and then incubated with the secondary
 antibodies anti-rabbit horseradish peroxidase (Jackson Immuno Research
 Laboratories, Inc., West Grove, PA, USA) and anti-mouse horseradish
 peroxidase (Jackson Immuno Research Laboratories, Inc.) at 1:2,000
 dilution and 25°C for 2 h. The PVDF membrane was washed five times with
 TBST, and the protein bands were visualized using a hypersensitive
 chemiluminescence kit (Boster). The PVDF membrane with bound antibodies
 was transferred into a plastic bag in a darkroom for photographing,
 developing, and fixing.
description: See README file.