| Penaeus monodon is an important aquaculture economic species in China.In recent years,the problem of shrimp pathogen infection caused by aquaculture environmental pollution has become increasingly serious,which restricts the healthy development of the shrimp culture industry.Vibrio is a type of Gram-negative pathogen commonly distributed in the estuary and coastal waters of the Gulf.Vibrio parahaemolyticus can cause diseases such as "red leg disease" and "rotten disease" of shrimp.The cause of "acute hepatic pancreatic necrosis syndrome",also known as "early death syndrome"(EMS),is the leading cause of large numbers of shrimp deaths.The innate immunity of shrimp is an important barrier to the health of shrimp.Therefore,in-depth study of the innate immune defense of P.monodon has important significance for controlling shrimp disease and healthy development of shrimp culture.Antimicrobial peptides(AMP)refer to a class of alkaline peptide substances with antibacterial activity produced in living organisms.They are found in all innate organisms and are an important part of the innate immune system.The antibacterial peptides found in crustaceans mainly include anti-lipopolysaccharide factor(ALF),crustin and penaeidin.Akirin is a highly conserved nuclear protein consisting of 180-204 amino acid acid residues and plays an important role in the regulation of innate immunity.Akirin is widely found in invertebrates and vertebrates.The study found that this gene plays an important role in the natural immune pathway of Drosophila.When it was silenced,the expression of the Drosophila antibacterial peptide Attacin was reduced by 90%.At the same time,fruit flies are clearly susceptible to Gram-negative bacterial infections.In this study,the Akirin gene of P.monodon(named PmAkirin)was successfully cloned and its immune function was initially explored.The PmAkirin cDNA sequence is 1508 bp in length and includes an ORF of 642 bp encoding 213 amino acids,a 50 bp 5'-UTR and a 816 bp 3'-UTR.The protein molecular weight of PmAkirin was predicted to be 23.82 KDa by ExPASy software,and the theoretical isoelectric point was 9.05.According to the existing literature analysis,PmAkirin contains two nuclear localization signals,which are 26~30(KRRRC)and 78~81(KRRK).This is consistent with the Akirin2 sequence of most mammals.In addition,PmAkirin also contains an antibacterial domain,the DEFSN domain(positions 3 to 34).Through multiple sequence alignment analysis,Akirin is highly conserved,and the N-terminal and C-terminal conserved are relatively strong.In evolution,we found that Akirin is divided into vertebrate and no by analyzing the sequence of Akirin protein of different species.There are two major classes of vertebrates,of which vertebrates are divided into two sub-categories: Akirin1 and Akirin2.PmAkirin is one of the invertebrates,and has the closest evolutionary relationship with P.vannamei and P.sinensis,and has a close relationship with the vertebrate Akirin2.It was found by qRT-PCR that PmAkirin was expressed in seven tissues including sputum,hepatopancreas,stomach,heart,intestine,muscle and hemolymph.Among them,PmAkirin has the highest expression in hemolymph,followed by heart,sputum,stomach and hepatopancreas,and low expression in intestine and muscle.In order to investigate the regulation of PmAkirin on the expression of antimicrobial peptides and its innate immunity in P.monodon,we performed interference experiments and overexpression experiments of dsRNA-Akirin and mRNA-Akirin.In situ hybridization experiments and qRT-PCR assays revealed that the interference and overexpression experiments of dsRNA-Akirin and mRNA-Akirin successfully interfered with and overexpressed PmAkirin.The PmAkirin mRNA expression level in hepatopancreatic tissue was significantly up-regulated after V.parahaemolyticus infected P.monodon.Compared with before infection,the mRNA-Akirin,dsRNA-Akirin,dsRNA-GFP,PBS and blank control groups were 1.67,1.74,1.75,1.5,1.74 times before infection,respectively,12 h after infection.This indicates that PmAkirin plays an important role in immune regulation.After interference with dsRNA-Akirin,the PmAkirin mRNA was specifically interfered for 24 hours,and then V.parahaemolyticus was injected,compared with the V.parahaemolyticus stimulation group,the mRNA levels of anti-lipopolysaccharide(ALF)PmALF2,PmALF3 and PmALF6 were 0.5,0.39 and 0.42,respectively,after interference with PmAkirin mRNA for 24 h.The expression levels of antibacterial peptide genes such as Crustin PmCrus1 and PmCrus4 were 0.39 and 0.35 times,respectively.The expression levels of antibacterial peptide genes such as Penaeidin PmPEN3 a,PmPEN3b and PmPEN5 were 0.44,0.45 and 0.46 times,respectively.After interference with mRNA-Akirin,the PmAkirin mRNA was specifically interfered for 24 hours,and then V.parahaemolyticus was injected,compared with the V.parahaemolyticus stimulation group,the expression levels of anti-lipopolysaccharide(ALF)PmALF2,PmALF3 and PmALF6 mRNAs were 1.7 and 1.74,respectively,after overexpression of PmAkirin mRNA for 24 h.2.61 times,the expression levels of antibacterial peptide genes such as Crustin PmCrus1 and PmCrus4 were 1.97 and 1.58 times,respectively,and the expression levels of antibacterial peptide genes such as Penaeidin PmPEN3 a,PmPEN3b and PmPEN5 were 1.86,1.79 and 2.07 times.This suggests that PmAkirin may be involved in the regulation of the expression of the antimicrobial peptide gene.Through the interference and overexpression experiments of dsRNA-Akirin and mRNA-Akirin,specific interference and overexpression of PmAkirin mRNA,followed by intramuscular injection of V.parahaemolyticus to infect P.monodon,and found dsRNA-Akirin interference group,shrimp hemolymph The concentration of V.parahaemolyticus was significantly higher than that of the blank control group,dsRNA-GFP group and PBS group,which was about 1.87 times that of the blank control group,while the survival number was far lower than the blank control group,dsRNA-GFP group and PBS group;In the mRNA-Akirin overexpression group,the concentration of V.parahaemolyticus in the hemolymph of shrimp was significantly lower than that of the blank control group,dsRNA-GFP group and PBS group,which was about 0.41 times of the blank control group,and the number of surviving was much higher than the blank control group,dsRNA-GFP group and PBS group.We speculate that PmAkirin participates in the clearance process of V.parahaemolyticus in the hemolymph of P.monodon by regulating the expression of antimicrobial peptides,thus affecting the survival rate of shrimp after pathogen infection.Through the interference and overexpression experiments of dsRNA-Akirin and mRNA-Akirin,specific interference and overexpression of PmAkirin mRNA,followed by intramuscular injection of V.parahaemolyticus to infect P.monodon,found(dsRNA-Akirin + V.parahaemolyticus)group,shrimp The concentration of V.parahaemolyticus in hemolymph was significantly higher than that in the bacterial infection group,(dsRNA-GFP+ V.parahaemolyticus)group and(PBS+ V.parahaemolyticus)group,which was about 1.87 times that of the bacterial infection group,and the survival number was far lower than the bacterial infection.Group,(dsRNA-GFP+ V.parahaemolyticus)group and(PBS+ V.parahaemolyticus)group;(mRNA-Akirin+ V.parahaemolyticus)group,the concentration of Vibrio parahaemolyticus in the hemolymph of shrimp was significantly lower than that of the bacterial infection group,(dsRNA-GFP+ V.parahaemolyticus)group And(PBS + V.parahaemolyticus)group,about 0.41 times of the bacterial infection group,the number of surviving is much higher than the bacterial infection group,(dsRNA-GFP + V.parahaemolyticus)group and(PBS + V.parahaemolyticus)group,we speculate that PmAkirin regulates the expression of antimicrobial peptides Participated in the removal process of V.parahaemolyticus in the hemolymph of P.monodon,which affected the survival rate of shrimp after pathogen infection.In this paper,the immune function of Akirin gene of P.monodon was studied.It was proved that PmAkirin can regulate the expression of downstream antimicrobial peptides.PmAkirin has a DEFSN antibacterial domain.These results indicate that PmAkirin plays an important role in the innate immunity of P.monodon.This provides a theoretical basis for in-depth study of the innate immune mechanism of shrimp and the promotion of healthy aquaculture. |
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