Molecular cloning, expression, and in situ hybridization analysis of MnGPx-3 and MnGPx-4 from oriental river prawn, Macrobrachium nipponense, in response to hypoxia and reoxygenation.

Lei Xu,Hongtuo Fu,Yiwei Xiong,Yongsheng Gong,Wenyi Zhang,Ming Yang,Hui Qiao,Shubo Jin,Yan Wu,Shengming Sun,Sufei Jiang

doi:10.1371/journal.pone.0229171

Lei Xu, Hongtuo Fu + Show 9 more

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https://doi.org/10.1371/journal.pone.0229171

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Abstract

Glutathione peroxidase (GPx) has been the focus of increased research because of its important role as an antioxidant and in reactive oxygen species (ROS) induced damage repair. Studies on GPxs have relevance with Macrobrachium nipponense because it has poor tolerance to hypoxia in Macrobrachium nipponense. The two subunits named as MnGPx-3 and MnGPx-4 according to the glutathione peroxidase nomenclature system. Both full-length cDNAs were cloned from the hepatopancreas. In this study, we analyzed the expression of two GPxs in Macrobrachium nipponense in response to changes in environmental oxygen. Expression levels of MnGPx-3 and MnGPx-4 indicated that both have strong responses to hypoxia. In situ hybridization showed that MnGPx-3 and MnGPx-4 were located in secretory and storage cells in hepatopancreas. These results suggest that GPx gene is expressed and released by secretory cells and released response to hypoxia. In the gill tissue, however, GPxs are located in blood cells, suggesting that they perform different functions in different tissues or organs. The results of in situ hybridization were consistent with those of quantitative Real-time PCR. This study provides a basis for understanding the oxidative stress response in M. nipponense under hypoxia.

Highlights

MethodsThis research was approved by the Institutional Animal Care and Use Ethics Committee of Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture, Zhejiang Institute of Freshwater Fisheries
The ternary catalytic center composed of Gln, Trp and Asn indicates that the Glutathione peroxidase (GPx) family has another catalytic mode, which is different from the traditional ternary catalysis and quaternary catalysis
A recent phylogenetic analysis indicated that the arthropod GPx homologues characterized to date form a clade with mammalian GPx3 [35], which is extracellular with broad substrate specificity [36]

Summary

Methods

This research was approved by the Institutional Animal Care and Use Ethics Committee of Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture, Zhejiang Institute of Freshwater Fisheries. In accordance with previous studies, all prawns were raised in six 500 L tanks at the appropriate parameters in a laboratory with aerated water for a week to adapt to the new environment [24,25]. 300 prawns were randomly divided into two groups (normal oxygen and hypoxia), and each group was set with three biological repeats. The oxygen concentration of the normoxic group was maintained at 6.0 ± 0.2 mg O2 L-1, whereas in the hypoxic group it was maintained at 2.0±0.2 mg O2 L-1 by bubbling with N2 gas [23]. After the challenge with hypoxia, normal oxygen conditions were restored. The samples were stored in ultra-low temperature freezer for the steps (ThermoFisher, USA)

Results

Discussion

Conclusion