Molecular Cloning and Expression of MnGST-1 and MnGST-2 from Oriental River Prawn, Macrobrachium nipponense, in Response to Hypoxia and Reoxygenation

Abstract

The glutathione-S-transferase (GST) superfamily includes seven classes, and different classes have different functions. GST superfamily members function in various processes including detoxification of xenobiotics, protection against oxidative damage, and intracellular transport of hormones, endogenous metabolites, and exogenous chemicals. Herein, to elucidate the tissue-specific expression pattern of GSTs in response to hypoxia stress, which induces cell death, we investigated the expression of GSTs in response to hypoxia and reoxygenation in oriental river prawn, Macrobrachium nipponense. Full-length cDNAs of two δ class GSTs were cloned from the hepatopancreas, and named MnGST-1 and MnGST-2 based on the established GST nomenclature system. Expression profiles of both GSTs in various tissues were different under acute and chronic experimental hypoxia stress conditions, suggesting that both respond strongly to hypoxia-induced oxidative stress. However, the intensity of responses to hypoxia and reoxygenation were different in different tissues. During acute hypoxia stress, MnGST-1 responds earlier than MnGST-2 in the hepatopancreas and gill, but more slowly in muscle. By contrast, during chronic hypoxia stress, MnGST-2 plays a more important role in the hepatopancreas and gill than MnGST-1.

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

“…A partial fragment of opsin cDNA was obtained from transcriptomic cDNA library of the M. nipponense hepatopancreas (NCBI, SRA051767.2) created in our lab. The isolation of total RNA and the synthesis of first-strand cDNA and 3'-rapid amplification of cDNA ends (RACE) was performed using relevant kits [1]. All primers for these steps are listed in Table 1.…”

“…NCBI related online analysis software was used to complete the deduction of amino acid sequence, protein analysis and nucleotide sequence analysis according to the previous method of our laboratory [1]. Multi-sequence alignment and phylogenetic tree construction were completed with DNAMAN 6.0 and MEGA 5.1, respectively.…”

“…The mRNA expression levels of the two MnGPx subunits following different treatments were measured by QPCR. Synthesis of the cDNAs and subsequent QPCR steps were completed as described previously [1,26]. According to previous laboratory studies, eukaryotic translation initiation factor 5A (EIF) was chosen as the reference gene of the relative quantification [27][28].…”

“…Dissolved oxygen (DO) has been widely accepted as the main indicator of water quality in the aquaculture of prawns [1]. When organisms are adversely stimulated by the external environment, the balance between oxidation and the antioxidant system is disrupted.…”

“…Previous evidence indicates that ROS are formed by electrons that accumulate during hypoxia [4], and acute reoxygenation after hypoxic treatment can also lead to the formation of ROS [5]. Excess ROS can cause changes in the structure and activity of many active substances, eventually leading to oxidative and metabolic damage [1,6]. To prevent damage from ROS, cells have developed a set of antioxidant defense systems, including glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), to maintain the redox balance and prevent oxidative damage [7].…”