[논문]Characterization and gene expression of heat shock protein 90 in marine crab Charybdis japonica following bisphenol A and 4-nonylphenol exposures

Park, Kiyun; Kwak, Ihn-Sil

doi:10.5620/eht.2014.29.e2014002

Characterization and gene expression of heat shock protein 90 in marine crab Charybdis japonica following bisphenol A and 4-nonylphenol exposures 원문보기

Environmental health and toxicology : Eht, v.29, 2014년, pp.2.1 - 2.7

Park, Kiyun (Department of Fisheries and Ocean Science, Chonnam National University) , Kwak, Ihn-Sil (Department of Fisheries and Ocean Science, Chonnam National University)

Abstract ▼ AI-Helper

Objectives Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone important in the maturation of a broad spectrum of protein. In this study, an HSP90 gene was isolated from Asian paddle crab, Charybdis japonica, as a bio-indicator to monitor the marine ecosystem. Methods This work reports the responses of C. japonica HSP90 mRNA expression to cellular stress by endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 4-nonylphenol (NP) using real-time. reverse transcription polymerase chain reaction. Results The deduced amino acid sequence of HSP90 from C. japonica shared a high degree of homology with their homologues in other species. In a phylogenetic analysis, C. japonica HSP90 is evolutionally related with an ortholog of the other crustacean species. The expression of HSP90 gene was almost distributed in all the examined tissues of the C. japonica crab but expression levels varied among the different body parts of the crabs. We examined HSP90 mRNA expression pattern in C. japonica crabs exposed to EDCs for various exposure times. The expression of HSP90 transcripts was significantly increased in C. japonica crabs exposed to BPA and NP at different concentrations for 12, 24, 48 and 96 hours. The mRNA expression of HSP90 gene was significantly induced in a concentration- and time-dependent manner after BPA or NP exposures for 96 hours. Conclusions Taken together, expression analysis of Asian paddle crab HSP90 gene provided useful molecular information about crab responses in stress conditions and potential ways to monitor the EDCs stressors in marine environments.

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제안 방법

Quantitative RT-PCR amplifications and measurements were carried out using an AB 7300 Real-Time PCR System (Applied Biosystems). The PCR for HSP90 was performed with 25 μL of a mixture containing 1 µL of cDNA template, 0.
The experiment was conducted in triplicate and the data are expressed as mean±deviation.

대상 데이터

Expression of heat shock protein 90 (HSP90) gene in various tissues of a Charybdis japonica crab. Ovary, gill, heart, stomach, hepatopancreas, eyestalk and muscles were collected from 10 crabs. beta-actin gene was used as an internal control to calibrate the cDNA template for all samples.
610). The network analysis consisted of at least three replicates.

데이터처리

Expression levels of HSP90 gene were normalized against the level of beta-actin using standard curves. Data of the fold change of HSP90 mRNA were logarithmically transformed before subjected to a one-way analysis of variance (ANOVA) using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA). When overall differences were significant, Tukey’s test was conducted to compare the means between individual treatments.

이론/모형

001. Phylogenetic tree of C. japonica HSP90 protein were made using the neighbor-joining method of MEGA 4.0.
The cycle threshold (Ct) was identified using the AB 7300 System SDS program. The expression levels were normalized against the beta-actin expression using the Ct method. Each experiment was calculated in triplicate.
For HSP90, this mixture was subjected to 33 cycles of amplification (denaturation for 30s at 94°C, annealing for 40s at 55℃ and extension for 50s at 72℃). The quality of the amplification was identified using an AB 7300 Real-Time PCR instruments (Applied Biosystems) to carry out dissociation curve analysis. The cycle threshold (Ct) was identified using the AB 7300 System SDS program.

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