| Antimicrobial peptides(AMPs)are an important part of the body’s innate immune system and have a wide range of inhibitory effects on pathogens such as bacteria,fungi and viruses.Shellfish is a filter-feeding aquatic organism,which is always faced with a large number of microorganisms in the water body.Therefore,shellfish has evolved an effective innate immune system,especially relying on its antibacterial peptides for immune defense.Pinctada fucata martensii is a kind of pearl oyster with important economic value,but currently no representative antimicrobial peptide molecule has been found in the mother oyster,which means that the mother oyster may rely on other immune effector molecules for immunity defense.In order to further explore the molecular mechanism of P.f.martensii against environmental pathogens,this study is based on the P.f.martensii genome database.From the genetic level,the immune effector molecules of P.f.martensii were screened and cloned.The tissue expression of immune effector molecules after challenged by Pathogen associated molecular pattern(PAMPs)was analyzed,prokaryotic expression vectors were constructed,and the antibacterial mechanism of recombinant proteins was studied.The results are as follows:(1)Screening and cloning of immune effector molecules: The local database was constructed using the online antimicrobial peptide database(APD)antimicrobial peptide amino acid sequence and compared with the P.f.martensii genome.Four immune effector molecules were successfully screened and cloned to obtain full-length c DNA,namely trypsin-like serine protease(PmTLS),Kunitz-type serine protease inhibitor(PmKuPI),goose-type lysozyme(Pmlys G)and saposin B(PmSap B).PmTLS c DNA is 778 bp full length,encoding 86 amino acids;PmKuPI c DNA is 1318 bp full length,encoding 398 amino acids;Pmlys G c DNA is 973 bp full length,encoding 255 amino acids;PmSap B c DNA is 1148 bp full length,encoding 214 amino acids.In homology analysis,the similarity between PmTLS,PmKuPI and PmSap B is low,while the similarity of Pmlys G is high,and the similarity with Argopecten irradians reaches 60.5%.Phylogenetic tree analysis revealed that PmTLS had the closest relationship with trypsin-like serine protease of Crassostrea gigas,PmKuPI had the closest relationship with Kunitz-type serine protease inhibitor of C.gigas and Crassostrea virginica,Pmlys G had the closest relationship with goose-type lysozyme of A.irradians,Azumapecten farreri and Mizuhopecten yessoensis,and PmSap B had the closest relationship with saposin of C.virginica.(2)Analysis of tissue differential expression and specific tissue expression after challenge of PAMPs: Real-time fluorescence quantitative PCR technology was used to detect the normal tissue expression pattern of four immune effector molecules,and it was found that PmTLS was expressed the highest in blood cells,PmKuPI was specifically highly expressed in the mantle,and Pmlys G and PmSap B were specifically highly expressed in the hepatopancreas.The three highly expressed tissues are all immune-related tissues.Compared with the control group,after LPS,PGN and Poly I:C challenge P.f.martensii,the expression of PmTLS in blood cells increased and reached the maximum expression level at 48 h.After LPS and Poly I:C challenge P.f.martensii,the expression of Pmlys G in the hepatopancreas reached the maximum value at 72 h(P <0.01),and after the PGN challenge,the expression of Pmlys G in the hepatopancreas reached the maximum expression level at 6 h(P <0.01).Similarly,after LPS and Poly I:C challenges,the expression of PmSap B in the hepatopancreas reached the maximum value at 72 h(P <0.01).After the challenge of PGN,the expression of PmSap B in the hepatopancreas reached the maximum expression level at 24 h(P <0.05).(3)Prokaryotic expression of four immune effector molecules: Successfully constructed four prokaryotic expression vectors pET28-PmTLS,pET28-PmKuPI,pET28-Pmlys G and pET28-PmSap B,optimized expression conditions,obtained recombinant proteins r PmTLS,r PmKuPI,r Pmlys G and r PmSap B in E.coli BL21(DE3).Under the induction of 0.5 m M IPTG,the optimal induction temperature of pET28-PmTLS was 37℃,and the optimal induction temperature of the remaining three expression vectors was 15℃.After purification and refolding,r PmTLS 8.02 mg,r PmKuPI 11.27 mg,r Pmlys G 16.96 mg,and r PmSap B 4.57 mg were finally obtained.(4)The antibacterial function of PmTLS and PmKuPI recombinant proteins(r PmTLS and r PmKuPI): The antibacterial activity of r PmTLS and r PmKuPI was detected by bacterial liquid growth inhibition method.Among the 8 bacteria tested,r PmTLS significantly inhibited the growth of four Gram-negative bacteria(P<0.05),namely Pseudomonas aeruginosa,Aeromonas hydrophila,Vibrio parahaemolyticus and Vibrio harveyi.r PmKuPI significantly inhibited the growth of five Gram-negative bacterial species(P<0.05),namely Escherichia coli,P.aeruginosa,A.hydrophila,V.parahaemolyticus and V.harveyi.The transmission electron microscope was used to observe the morphological changes of bacteria with two recombinant proteins.r PmTLS was found to act on P.aeruginosa,A.hydrophila and V.parahaemolyticus.Compared with the control group,the bacterial cells of P.aeruginosa swelled,the internal structure was significantly changed,and the phenomenon of plasmolysis occurred.The contents of A.hydrophila are partially lost,the internal structure changes,and the cell wall dissolves.The content of V.parahaemolyticus is missing,the middle of the bacteria swells,and the phenomenon of plasmolysis occurs.r PmKuPI acts on P.aeruginosa and V.parahaemolyticus.Compared with the control group,the content of P.aeruginosa was missing,the internal structure was significantly changed,and the content of the bacteria was also released at one end.The contents of V.parahaemolyticus contracted,basically detached from the cell wall,and there were obvious folds and deletions.This study provides a preliminary reference for exploring the mechanism of action of P.f.martensii immune effector molecules,further understanding of the mechanism of shellfish resistance to environmental pathogens,and the possibility of alternative antibiotic abuse in aquaculture. |
