| Antimicrobial peptides(AMPs)are small molecular proteins with antibacterial activities and important physiological significance and important application potential.Mussel AMPs are an important research field for minging novel peptides with biological antimicrobial activities.So far,Mytilus has become an important species in the study of AMPs in marine organisms because of the strong antibacterial activity and abundant molecular diversity of AMPs in this genus.Mytilus coruscus is one of the most extensive mussel species distributed along the Western Pacific and also the most important cultured shellfish in the East China Sea,indicating that the M.coruscus has both of important economic and research values.Previous studies revealed that Mytilus showed strong environmental adaptability,in which,the mussel AMPs may play important roles in mussel inmmunlogy,and also have great significance for the immune defense of M.coruscus.Abundant AMPs,including Mytichitins and Myticusins,identified previously from this species.However,the molecular diversity and expression pattern of M.coruscus AMPs remain largely unknown.Based on the dataset of M.coruscus hemocyte transcriptome,we identified twelve AMPs representing the main AMPs families of M.coruscus,including three novel mussel AMPs(Arthropod-like defensin-1,Arthropod-like defensin-2 and Crustin-like).The expression profiles of these AMP genes have been analyzed in different tissues,different larval development stages,and different individuals of M.coruscus,as well as in hemocytes and gonads under the threat of different microbes.The results suggested that in adult M.coruscus,the gonads and hemocytes are the main sources of AMPs for the mussels collected in winter and summer,respectively.In addition,four antimicrobial peptides showed higher expression level in gonad of the male mussels than that in the female mussels,indicating that these four antimicrobial peptides may have a special protective effect on male mussels.The expression of AMPs is developmentally regulated,and most AMPs are undetectable in larvae until after the larval settlement and metamorphosis.In vivo microbial challenges significantly increased the expression of AMPs in M.coruscus hemocytes,and different AMP response patterns under challenges from different microbes were observed,showing a rapid,strong response to gram-positive strains,a weak response to gram-negative strains,and a long-term response to fungus.While the gonads showed a different response pattern comparing with the hemocytes,with stronger changes in expression relative to the control and stronger fluctuation in the expression level of some AMPs.Finally,LC-MS/MS was used for peptidomic identification of the AMPs in M.coruscus,and a total of four kinds of AMPs were identified from mussel serum,indicating that mussel AMPs have complex expression characteristics from gene to peptidomic.For further exploring the molecular characteristics of AMPs in M.coruscus,myticusin-1 was selected as the research object.The recombinant expression of Myticusin-1 was performed using Escherichia coli expression system combined with codon optimization strategy.After renaturation and purification,the recombinant Myticusin-1 showed broad spectrum antibacterial activity against Gram-positive bacteria,Gram-negative bacteria and fungi.Among which,the recombinant Myticusin-1 showed strong antimicrobial activities against Gram-positive bacteria.The successful expression of myticusin laid a foundation for the subsequent development of molecular resources of AMPs in M.coruscus.The study from the level of gene expression and protein for the first time to complete the molecular diversity and functional analysis of the AMPs in mussels,and also preliminarily carried out the prokaryotic recombinant expression attempt of AMPs in M.coruscus.The above research laid a foundation for the follow-up in-depth understanding of the disease tolerance mechanism of mussels and the peptidomic engineering and resource development based on AMPs in mussels. |
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