Molecular Cloning And Functional Analysis Of The Crustacean Antimicrobial Peptides And STAT (Signal Transducer And Activator Of Transcription)

The Crustacea is the largest group of aquatic arthropods. The cultivation and marine fishing of crustaceans are conspicuously important activities in the world nowadays. However, the aquaculture is suffering serious loss every year because of the outbreaks of diseases caused by virus and bacteria. Due to the huge commercial importance, it is essentially to comprehend the immune responses of crustaceans to control the disease outbreaks.In this study, the members of two crustacean antimicrobial peptide families, crustins and anti-lipopolysaccharide factors, were investigated to understand their biological activities and functions. Besides, the signal transducer and activator of transcription (STAT) was cloned from the Chinese white shrimp, Fenneropenaeus chinensis, and its expression patterns were analyzed.1. Characterization of crustins from the Chinese white shrimp, Fenneropenaeus chinensisThree members of antimicrobial peptides crustins (designated Fc-crus 1, Fc-crus 2, and Fc-crus 3) were obtained by the previous study of our lab. In this study, its cDNA and deduced amino acids sequences were analyzed. It was revealed that the deduced peptides of Fc-crus 2 and 3 contain a signal peptide and a crustin domain at the C-terminal formed by twelve conserved cysteine residues. The partial sequence of Fc-cru 1 contains a crustin domain as well. Phylogenetic analysis revealed that Fc-crus 1 and Fc-crus 2 belong to the crustins cluster; while the Fc-crus 3 was classified into the carcinins group, which is surprisingly because Fc-crus 3 is, as we know, the first carcinin-like protein found in penaeid shrimp. The expression of Fc-crus 1 and Fc-crus 2 was detected in each developmental stages, whereas the mRNA of Fc-crus 3 was not detected in the developmental stages extending from nauplii, mysis and post larvae to ovaries of juvenile shrimp. The recombinant proteins containing mature Fc-crus 2 and Fc-crus 3 were recombinantly expressed in E. coli and purified respectively. The antibacterial assays revealed that the recombinant mFc-crus 2 could inhibit the growth of Gram-positive bacteria in vitro.2. Characterization of anti-lipopolysaccharide factors from red swamp crayfish, Procambarus clarkiiTwo anti-lipopolysaccharide factors (ALFs) cDNA sequence(PcALF1 and PcALF2) were identified from red swamp crayfish, Procambarus clarkii. The deduced peptides of the two PcALFs are conserved; they manifested the signal peptide and lipopolysaccharide (LPS)-binding domain, especially the two conserved cysteine residues at both ends of the domain. Transcripts of PcALF1 and PcALF2 were detected in multiple tissues. Results of quantitative real-time PCR exhibited that the expression levels of PcALFl and PcALF2 were induced by the challeng of virus, Gram-positive and Gram-negative bacteria. Purified recombinant protein of PcALFl revealed multiple biological activities:it gave all the tested bacteria and fungi a tight binding; it could bind microbial polysaccharides (LPS, LTA, and?-glucan) as well. In vitro, the antimicrobial activity assay was demonstrated as a broad spectrum against Gram-positive and Gram-negative bacteria and a fungus. The rPcALF1 also exhibited a clearance activity on Vibrio anguillarum in a dose-dependent manner in vivo.3. Molecular cloning and expression analysis of signal transducer and activator of transcription (STAT) from the Chinese white shrimp Fenneropenaeus chinensisInnate immunity is the first line of defense by a host against invading pathogens. Several signaling pathways participate in the immune response, one of which is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. Various evidences have been provided to suggest that the JAK/STAT pathway is involved in both antibacterial and antiviral immunities. In this study, the full-length cDNA and gene sequence of STAT (designated as FcSTAT) was cloned from the Chinese white shrimp, Fenneropenaeus chinensis. Phylogenetic analysis reveals that the FcSTAT is clustered with STAT5s and STAT6s from vertebrates and STATs from invertebrates. Quantitative real-time PCR exhibited that the FcSTAT had a wide distribution in all detected tissues and developmental stages. Time course analysis of the transcription level after WSSV challenge showed a noticeably early up-regulation of FcSTAT in hemocytes, hepatopancreas, and intestines. The expression levels of FcSTAT increased corresponding to Vibrio anguillarum stimulation in both hemocytes and hepatopancreas as well. All these imply that the JAK/STAT pathway participates in the immune response against bacteria and virus in F. chinensis.