Cloning,expression And Anti-Vibrio Parahemolyticus Of A Novel Antimicrobial Peptide ALF-like Gene From Penaeus Monodon

As one of the three major aquaculture shrimp in the world,Penaeus monodon is an inherent breed in our country,and its aquaculture industry occupies an important position in the world aquaculture industry.In recent years,with the emergence and development of high-density and intensive farming mode,the breeding environment deteriorates and diseases occur frequently.However,due to the lack of acquired immune pathway,the innate immune pathway is the main defense against the invasion of external pathogens.Antimicrobial peptide(AMP)has been proven to be an important effector in innate immunity and has a broad antibacterial effect.Anti-lipopolysaccharide factor(ALF)is a class of antimicrobial peptide molecules mainly present in crustaceans,which is regarded as a promising new generation of antimicrobial agents and plays an important role in the innate immunity of prawn.Therefore,a series of studies on ALF genes and elucidating the disease-resistance mechanism can not only provide scientific data for disease prevention and control means or product development in the process of shrimp breeding,but also be of great significance for scientific development planning of Chinese green and healthy aquaculture industry.In this study,we cloned the c DNA sequence of antibacterial peptide PmALF-like gene(PmALF-like)of P.monodon,identified its expression rule under stimulation by Vibrio parahaemolyticus,and verified its antibacterial activity in vitro.And the antibacterial effect of P.monodon on V.parahaemolyticus was studied.The specific experimental results are as follows:(1)The c DNA sequence of PmALF-like gene was obtained by RACE,and its sequence characteristics and evolutionary status were analyzed,and its protein structure was predicted.The results showed that the full length of PmALF-like gene c DNA sequence was 1242 bp.It includes the 5’ Untranslated Regions(UTR)of 45 bp,the 3’UTR of 798 bp and the Open Reading Frame(ORF)of 399 bp to encode 132 amino acids.The molecular weight was 15.26 k Da and the theoretical isoelectric point(p I)was 7.67.The signal peptide of PmALF-like protein was predicted by the online software Signal P-5.0 and the signal peptide splicing site was found at the 18 th amino acid.The amino acid sequence of PmALF-like was similar to that of ALF in other species,among which the similarity was high with that of ALF-like in Litopenaeus vannamei,ALF-H1 in Penaeus japonicus,and ALF-like in Penaeus chinensis.The phylogenetic evolutionary tree based on the similarity of ALF amino acid sequences showed that the PmALF-like amino acid sequences first clustered into a branch with L.vannamei,and then clustered into a large branch with P.japonicus and P.chinensis.(2)Real-time fluorescence quantitative PCR(qRT-PCR)was used to analyze the tissue expression of PmALF-like gene and its expression characteristics under the stimulation of V.parahemolyticus.The results showed that PmALF-like gene was expressed in different tissues of P.monodon,and the expression abundance was highest in hepatopancreas,followed by hemolymph,intestine,heart,gill and muscle.At 6 h,12 h,24 h and 48 h after injection of V.parahaemolyticus,the expression level of PmALF-like gene in hemolymph tissue of penaeus penaei was significantly higher than that of blank group.At 6 h,12 h,24 h,48 h and 72 h after injection of V.parahaemolyticus,the expression of PmALF-like gene in hepatopancreas was significantly higher than that in blank group.In situ hybridization results showed that the positive signal of PmALF-like gene in hepatopancreas of P.monodon at 6 h and12 h after injection of V.parahaemolyticus was significantly stronger than that in PBS group,which further indicated that the stimulation of V.parahaemolyticus could significantly change the expression of PmALF-like gene at the subcellular level.Injection experiments showed that PmALF-like recombinant protein could reduce the death of P.monodon after infection with V.parahaemolyticus.(3)PmALF-like peptide was obtained and the bacteriostatic function of the recombinant protein was analyzed in vitro.PmALF-like mature peptide without signal peptide was successfully obtained by prokaryotic expression method in vitro.The effects of PmALF-like peptides on Aeromonas veronii,V.Parahemolyticus and Aeromonas hydrophila were found by liquid medium method and Oxford Cup method The growth of hydrophila has obvious inhibitory effect.PmALF-like gene was overexpressed by injection of mature peptide PmALF-like,and it was found that after stimulation by V.parahemolyticus,the death number of P.monodon PmALF-like was significantly lower than that of control group,indicating that PmALF-like could effectively resist pathogen invasion and improve the survival rate.(4)In order to further study the functional mechanism of PmALF-like against the invasion of V.parahemolyticus,the immunoenzyme activity of Alkaline Phosphatase(AKP),Acid Phosphatase(ACP),and Superoxide Dismutase(SOD)in the hepatopancreas and hemopoietic tissues of P.monodon has been measured and the results showed that the SOD,ACP and AKP activities of the control group were low and relatively stable.The SOD activity of PBS+V.P treatment group,Trx+V.P treatment group and PmALF-like+V.P treatment group was significantly increased at0-6 h(p < 0.05),and the SOD activity decreased at 6 h,24 h,48 h and 72 h with the increase of injection time.Compared with PBS+V.P and Trx+V.P,ACP and AKP in PmALF-like+V.P treatment group could reach the maximum faster.These results indicated that V.parahaemolyticus infection had significant effects on the activities of SOD,ACP and AKP in hepatopancreas and hemolymph tissue of P.monodon.Injection of mature peptide PmALF-like can appropriately reduce the number of death of P.monodon infected with V.parahaemolyticus,which may enhance the innate immunity of P.monodon.