Three Peroxiredoxins From Penaeus Monodon:Identification,Characterization,Functional Analysis Under Biotic And Abiotic Stresses

Penaeus monodon,as one of three most commercially important hatchery species in the world,is much beloved due to its advantages over other in terms of its high growth rate,size,unique taste,high nutritive value,salinity tolerance,high price and persistent demand in international markets.But,so far there are two main problems for P.monodon aquaculture that the first one is disease prevention and the second is the breeding of good breeding.The black tiger shrimp,lacking a specific immune response and immunological memory,rely on the innate immune system in facing with diseases.Innate immunity at an early stage is the first line of defense that assists to restrict infection.Disease problems are still difficult to control.P.monodon is suffered from a series of environment stimuli,such as sheer salinity,pathogens.Peroxiredoxins?Prxs?are ubiquitous,multifunctional and evolutionarily conserved enzymes that can protect cells from oxidative damage caused by ROS and play a vital role in development,reproduction and immune responses to biotic and abiotic stresses at the molecular level.So,the aim of this work was to explore the function of peroxiredoxins superfamily member about immune responses.Then we identified,cloned and expressed three Prxs genes from P.monodon including PmPrx1,PmPrx5,PmPrxn to study peroxiredoxins superfamily in order to solve the prawn's present situation.In this study,the cDNA partial sequences of PmPrx1,PmPrx5 and PmPrxn were obtained from the transcriptome of P.monodon from our laboratory.By means of molecular biology techniques,the full length of PmPrx1,PmPrx5,PmPrxn genes was cloned.To study the potential of the three genes in P.monodon culture.First,qRT-PCR was performed on each tissue of P.monodon to initially understand the distribution of the three genes.After bacterial immune infection and different environmental toxicity stresses,the transcription level of the three genes in gill and hepatopancreas was detected.To determine whether it is involved in the immune defense process.Moreover,recombinant proteins of the three genes have been obtained and purified.The antioxidant activity of recombinant proteins was tested.In vitro the antibacterial activity was examined.The relationships between them and heavy metals and H₂O₂ were tested.The concrete research content is summarized as follows:?1?Here,a full-length Prx1 cDNA sequence?PmPrx1?was isolated from P.monodon.The PmPrx1 cDNA was 951 base pairs?bp?,encoding198 amino acid polypeptides.The results of qRT-PCR showed that the PmPrx1 mRNA was ubiquitously expressed in all tissues tested and had a comparatively high expression level in immune-associated tissues?gill,hepatopancreas?.To explore the immune and anti-stress roles of PmPrx1,the gills and hepatopancreas were chosen as target tissues in Penaeus monodon and were challenged with bacteria?Vibrio harveyi and Streptococcus agalactiae?and toxic environmental stresses.To further clarify the immune function of PmPrx1 after bacterial challenge,the recombinant PmPrx1 protein was acquired using a prokaryotic expression method.The antioxidant activity of the recombinant PmPrx1 was assessed by the catalyzing hydrogen peroxide assay method,and the results showed obviousantioxidantactivityinadose-dependentand temperature-dependent manner.The antimicrobial activity of purified PmPrx1 protein was evaluated and further studied in vitro relying on a bacterial growth inhibition test which was conducted in both liquid and solid cultures.Furthermore,E.coli transferred with pRSET-PmPrx1 was dramatically protected in response to metal toxicity and H₂O₂ oxidative stress.?2?As a unique atypical 2-Cys Peroxiredoxin?Prx?of the Prx-like superfamily,Peroxiredoxin5?Prx5?possesses special properties,such as its enzymatic mechanism,wide subcellular distribution and high affinity for peroxides and peroxynitrite.Prx5 plays a crucial role in oxidative stress,immune responses,cell apoptosis,proliferation,differentiation,intracellular signaling,the modulation of gene expression,ecdysis,etc.In this paper,we obtained a full-length Prx5 cDNA sequence?designated PmPrx5?from black tiger shrimp?P.monodon?.The full-length PmPrx5cDNA sequence was 1686 bp containing a 5'untranslated region?UTR?of76 bp with two nucleotide sequences?AAA?,a 3'UTR of 1040 bp with a poly?A?tail and two canonical polyadenylation signal sequences?AATAAA?,and an open reading frame of 570 bp encoding 189 amino acid residues with a predicted molecular mass of 20 kDa and a theoretical isoelectric point of 6.29.Phylogenetic trees and multiple sequence alignment showed that the PmPrx5 had strong homology with Prx5proteins from other species,such as similarity with Palaemon carinicauda?69%?and Macrobrachium rosenbergii?69%?,containing the highly conserved functional domain.PmPrx5 mRNA was ubiquitously detected in all tested tissues.After P.monodon was exposed to pathogenic bacteria,osmotic pressure,acidity and alkalinity and the heavy metal,the mRNA expression of PmPrx5 in the gills and hepatopancreas was significantly enhanced?P<0.01?because of the immune response and declined with heavy metal copper and cadmium challenges as time progressed.The recombinant PmPrx5 protein purified in E.coli?DE3?was further confirmed to exhibit antioxidant activity and antibacterial properties to a certain extent using a bacterial growth inhibition test in both liquid and solid cultures in vitro.E.coli transformed with pRSET-PmPrx5 were dramatically protected in response to metal toxicity stress.?3?PmPrxn was successfully identified,cloned and expressed relying on prokaryotic expression system.Tissue-specific expression analysis revealed that PmPrxn were ubiquitously expressed with the highest expression levels in the hepatopancreas,followed by gills and ovaries.Quantitative RT-PCR analysis exhibited that PmPrxn mRNA expressions were distinct when P.monodon suffered from biotic and abiotic stresses including Vibrio harveyi,Streptococcus agalactiae and toxic environmental stresses infection,indicating that the PmPrxn might play a vital role in response to biotic and abiotic stresses.Then,purified PmPrxn protein was evaluated and investigated in vitro to have antimicrobial property.Furthermore,DE3-pRSET-PmPrxn was observably protected against metal toxicity.In summary,this study provides useful information about the role of PmPrx1,PmPrx5,PmPrxn in defense against a variety of toxic factors in shrimps that help to further clarify the function of Prxs.