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.

Quantitative real-time PCR (qRT-PCR)

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^−ΔΔCt 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.

Western blot analysis 

Approximately 100 mg C. chinensis 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.

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