Expression and functional characterization of glutamine synthetase from giant freshwater prawn (Macrobrachium rosenbergii) under osmotic stress

Abstract

The freshwater prawn, Macrobrachium rosenbergii naturally lives in the freshwater, though it migrates to the brackish water environment during spawning that claimed to be resistant on a broad range of saline fluxes. However, little is known about the osmoregulatory patterns and the effect of an enzyme glutamine synthetase (GS) in M. rosenbergii under stress. Here, we described the identification and functional characterization of GS from M. rosenbergii (Mr-GS) at molecular and protein levels. The identified Mr-GS was comprised of 361 amino acids that phylogenetically shared the highest identity with other crustaceans and predicted to contain Gln-synt_C and Gln-synt_N domains at the respective terminal regions. Tissue distribution analysis in M. rosenbergii revealed that the Mr-GS was highly expressed in muscle, and commonly existed in other examined tissues in the following order gills > heart > stomach > brain > haemolymph. Whereas, the mRNA of Mr-GS was significantly up-regulated in the muscle and gill tissues following challenges with either hyper (0 → 13‰), or hypo (13 → 0‰) osmotic stress at 3, 6 and 12 hr. Furthermore, the level of Glutamine concentration was positively correlated with the GS mRNA and protein expression patterns in hyper-osmotic stress, whereas in hypo-osmotic stress a slight decrease in the gills and maintained a level in the muscle tissues at 3, 6 and 12 hr post-treatments. Our findings suggest that Mr-GS potentially exhibited the osmoregulation responses in the gill and muscle tissues of M. rosenbergii throughout the time of osmotic stress, which will benefit for future study on osmoregulation. K E Y W O R D S glutamine, glutamine synthetase, Macrobrachium rosenbergii, osmoregulation, osmotic stress How to cite this article: Lu Z, Qin Z, Sarath Babu V, et al. Expression and functional characterization of glutamine synthetase from giant freshwater prawn (Macrobrachium rosenbergii) under osmotic stress.

The Encystment-Related MicroRNAs and Its Regulation Molecular Mechanism in Pseudourostyla cristata Revealed by High Throughput Small RNA Sequencing

“…A previous report found that the GS gene has osmo-regulation properties in the osmotic stress environments. Moreover, GS participated in osmo-regulating the toxic ammonia and increased the Gln levels in the tissues [44]. Furthermore, the mRNA expression was significantly upregulated in the P. cristata during the osmotic challenge in the encystment.…”

Insight into the Regulatory Relationships between the Insulin-Like Androgenic Gland Hormone Gene and the Insulin-Like Androgenic Gland Hormone-binding Protein Gene in Giant Freshwater Prawns (Macrobrachium rosenbergii)

“…The RNA was isolated and RNase-free DNase I (Sangon, Shanghai, China) treatment was administered to remove any possible contamination by genomic DNA. Using a biophotometer (Eppendorf, Hamburg, Germany), the concentration of the total RNA in each sample was noted, after which 1% agarose gel was used per 2 μL for electrophoresis to evaluate all the sample’s integrity [ 42 ]. Each RNA had their first-strand cDNA developed to make real-time reverse transcription-quantitative polymerase chain reaction (qRT-PCR) possible; this was done with 1 μL of the total RNA, 4 μL of 5× iScript reaction mix (Bio-Rad, Hercules, CA, USA), and 1 μL iScript reverse transcriptase in a final volume of 20 μL.…”

“…The membranes were then rinsed for 5 min in three repetitions with TBST and also incubated with HRP-conjugated secondary antibody goat anti-rabbit IgG (1/10,000 dilution) for 55 min. Another three repeats for 5 min washing with TBST were done [ 42 ]. This led to the revealing and measurement of the immunoreactive bands by chemiluminescence (ECL Western Blotting Substrate, Solarbio, Beijing, China) and ChemiScope 6000 (CliNX, Shanghai, China), respectively.…”

“…The conditions provided were 95 °C for 3 min, 40 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 20 s, and finally 4 °C for 5 min. The ratio of expression in correlation of the target genes was standardized with an internal reference β-actin gene [ 42 , 43 ], and the 2 −ΔΔCT method was used to calculate expression levels of Mr - IAGBP and Mr - IAG [ 47 ].…”

Aeromonas Hydrophila Infection Activates Death Receptor Apoptosis Pathway in the RBCs of Grass Carp (Ctenopharyngodon idellus)

“…Whole formation was depicted as a mean ± SD (standard deviation) at least 3 separate tests. The statistical importance of the information was evaluated by ANOVA (one-way analysis of variance), and GraphPad Prism 7 was used to draw gures [31]. The difference was considered important when P < 0.05 (*).…”