| Scylla paramamosain is an important marine crab in China with high economic and nutritional value.However,in the process of artificial breeding,crabs are easily threatened by water quality and pathogenic microorganisms,which would affect their growth.Especially in the vulnerable larval stage,the extremely high mortality rate seriously hinders the development of the crab farming industry.Therefore,it is very important to study the innate immune function in the early developmental stage of crabs.The transcriptome data from previous experiments showed that some immune-related genes were significantly induced after artificial infection,including antimicrobial peptide,Toll and IMD signaling pathway,proPO system,complement system and coagulation system.A new Crustin homologous gene named SpCrus7 was screened from the transcriptome database of larvae infected with lipopolysaccharide(LPS)and Vibrio alginolyticus.Crustin is an antimicrobial peptide widely distributed in crustaceans and plays an important role in the process of crab resistance to pathogenic invasion.The expression characteristics of SpCrus7 gene in crab,the antimicrobial function and mechanism of its recombinant protein were studied.The main research results of this paper are as follows:1.The full-length cDNA sequences of SpCrus7 were cloned.The full-length of SpCrus7 cDNA sequence is 658 bp,encoding 109 amino acids(aa).Its mature peptide contains 88 aa and the relative molecular weight(MW)and isoelectric poin(pI)of which is 9.8 kDa and 8.49.The primary structure contains signal peptide,cysteine enrichment region and WAP domain,and the secondary structure is mainly composed of alpha helixes and beta foldings.It belongs to Type I Crustin.2.The mRNA expression characteristics of SpCrus7 in crabs at different developmental stages,tissues of adult crabs,and gonads before or after mating were revealed.The results showed that:(1)The transcription level of SpCrus7 increased gradually at the early stage of embryonic development;SpCrus7 gene expresstion increased first and then decreased in larval stage from zoea I to juvenile I stage,and it showed the highest expression in Zoea V stage than that of other larval stages.In male crabs,Spcrus7 gene expressed predominantly in penis,followed by eyestalk.(2)It’s worth noting that,the expression of SpCrus7 in the spermatheca and reproductive tract of crab was significantly higher than that of other tissues,the highest in the spermatheca,followed by the reproductive tract.(3)The mRNA level of SpCrus7 was significantly different between the spermatheca and the reproductive tract of female crabs before and after mating.It was significantly up-regulated after mating and maintained high expression level,suggesting that SpCrus7 may playe a role in the reproductive system of S.paramamosain.3.The expression pattern of SpCrus7 gene induced by Vibrio alginolyticus was revealed.SpCrus7 was significantly up-regulated at 12 h in the eyestalks of male crabs stimulated by Vibrio alginolyticus,6 h in the hemocytes of male crabs;but down-regulated at 12 h in the spermatheca of female crabs by V alginolyticus.All these results indicated that SpCrus7 may be involved in the immune response process of S.paramamosain.4.The recombinant protein of SpCrus7 mature peptide named pSpCrus7 was obtained and its in vitro activity was elucidated.The recombinant protein pSpCrus7 was expressed and purified by prokaryotic expression system and affinity chromatography.Though it exhibited no agglut:ination effect on the tested bacteria,pSpCrus7 had bactericidal activity against Micrococcus.luteus,M.lysodeikticus,Corynebacterium glutamicum,Staphylococcus aureus,Pseudomonas stutzeri and Shigella flexneri,and the minimum inhibitory concentration(MIC)are 24-48 μM,12-24 μM,12-24μM,24-48 μM,24-48 24-48 μM respectively,and it can also affect the growth of Bacillus subtilis.The bactericidal kinetics curve showed that pSpCrus7 could kill all the bacteria within 120 min after incubated with S.aureus and M.luteus,within 240 min with M.lysodeikticus and S.flexneri respectively.The antimicrobial activity of pSpCrus7 has poor thermal stability and is susceptible to Na+.5.The in vitro activity of synthetic peptide SpCrus722-42 was revealed.SpCrus722-42 has bactericidal effect on M.luteus,M.lysodeikticus,C.glutamicum,S.aureus,P.stutzeri and B.subtilis,and the MIC values are 12-24μM,12-24μM,12-24μM,12-24μM,24-48 μM and 24-48 μM respectively.The MIC values of SpCrus722-42 are less than or equal to those of pSpCrus7.The bactericidal kinetics curves showed that pSpCrus7 could kill all the bacteria within 30 min after incubated with S.aureus and C.glutamicum,within 15 min with M.luteusand M.lysodeikticus,respectively.The time of SpCrus722-42 needed to kill all the bacteria was shorter than that of pSpCrus7,which indicated that the antimicrobial activity of synthetic peptide SpCrus722-42 was stronger than that of pSpCrus7.The SpCrus 722-42 also had no agglutination effect on the tested bacteria,and the stability of the antimicrobial activity is susceptible to Na+but therm.In addtion,SpCrus722-42 exhibit no cytotoxicity against insect cells(Sf9,High Five),mammalian cells(293T,HepG2,HeLa)and haemocytes of S.paramamosain.6.The eukaryotic expression vector ppic9k-spcrus7 was constructed,and the mature peptide eSpCrus7 of SpCrus7 was obtained by eukaryotic expression system and affinity chromatography.The eSpCrus7 had bactericidal activity against M.luteus,M.lysodeikticus,C.glutamicum,S.aureus,P.stutzeri and S.flexneri,and the minimum inhibitory concentration(MIC)are 12-24 μM,12-24 μM,24-48 μM,24-48μM,24-48μM,24-48μM respectively,and it can also affect the growth of Bacillus subtilis.The MIC values of eSpCrus7 are higher than those of SpCrus722-42 and equal to pSpCrus7.7.The antimicrobial mechanism of SpCrus7 was preliminarily elucidated.Scanning electron microscopy(SEM)observation results showed that the cell integrity and microstructure of bacteria treated by SpCrus722-42 changed obviously,in which the bacteria were broken and the contents were discharged.The bacteria treated by SpCrus722-42 were counted by flow cytometry,and the permeability of bacterial cell membrane changed.Microbial surface polysaccharide binding experiments showed that pSpCrus7 could recognize and bind LPS,PGN,Glucan and LTA.It was found in microbial binding asassy that pSpCrus7 could bind to the tested bacteria.Confocal microscopy observation results showed that pSpCrus7 could be localized to the surface of the tested bacteria.The results showed that SpCrus7 might bind to the surface of bacteria,thereby destroying the integrity of cell membrane,causing the contents to flow out and the bacteria to rupture and die.However,the specific antimicrobial mechanism of SpCrus7 still needs to be further studied. |
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