Expressing And Functional Analysis Of SWD Antimicrobial Peptide, Glutamine Synthetase And Ras In Chinese Shrimp Fenneropenaeus Chinensis

Intensive aquaculture of crustaceans,especially of penaeid shrimp,is one of the fastest growing sectors in aquaculture production.Despite its huge success,shrimp culture is facing severe outbreaks of infectious diseases.During the past few years, commercial shrimp farming has been severely hit due to epidemics associated with viruses and vibriosis,which have caused serious economic losses.Disease outbreaks are resulting from interactions of the three factors of a deteriorating environment,weakened hosts,and aggressive pathogens.Crustaceans do not have acquired immunity;instead they have an innate immune system,which includes melanisation by activation of the prophenoloxidase-activating system(proPO system),a clotting process,phagocytosis,encapsulation' of foreign material, antimicrobial action,and cell agglutination.The control of diseases and biological security have been regarded as the key factors which affect the continuous and stabilizing development of penaeid shrimp culture.Enhanced the innate immune shrimp can increase resistance to infection of broad-spectrum.Activated innate immune system or increased the variety of immune-enhancing drug activity is generally considered to be a good choice for prawn breeding to improve the health and resist epidemic outbreaks of disease. Immune-enhancing drugs are the material that can stimulate the immune response and enhancing the immune system to resist infection and disease.Such asβ- glucan,can enhance crustaceans resistance parasites,bacteria and virus infection capacity.Antibacterial peptide from their own shrimp for disease prevention and control can not only enhance the innate immune shrimp,resist the invasion of pathogenic microorganisms,but also to ensure biosafety.So the research of the shrimp antibacterial peptide,in particular,which has a dual function of antibacterial peptide is of great significance.Antibacterial peptide of shrimp in the exercise of functions is not an independent body to complete the required immune system and immune-related signal transduction pathway of participation and the participation of other factors common to complete the function.The study of genes related to immune-related signal transduction mechanism,understanding the shrimp signal transduction pathway in the invasion of pathogenic microorganisms in the role of innate immunity is of great significance,thereby better understanding of pathogenic microorganisms shrimp resistance mechanisms.The presence of WAP domains within proteins has been suggested to confer either proteinase inhibitor or antibacterial activity on these molecules and the mechanism of immuno-related signal transduction is not extensively studied in shrimp as well as in marine invertebrates.The Fc-SWD,GS and Ras cDNAs were cloned in the studies.The charaters and functions of the three genes could help us know about the inmmune defence mechanisms and the signal transduction mechanisms.1.A single whey acidic protein domain(SWD)-containing peptide from the fleshy prawn with antimicrobial and proteinase inhibition activitiesShrimp rely upon an innate immune system to live in marine environments under a variety of stresses,including bacterial,fungal,and viral challenges.The production and release of antimicrobial peptides are an important immune response to fight these pathogens.A gene for a new type of antimicrobial peptide that contains a single whey acidic protein domain(SWD) was cloned from the haemocytes of fleshy prawns (Fenneropenaeus chinensis) and this peptide is named Fc-SWD.The full-length cDNA of Fc-SWD was 399 bp and contained a single open reading frame(ORF) of 273 bp,encoding an Fc-SWD of 90 amino acids,including a putative 24-amino acid signal peptide,and a mature 66 residue peptide.Sequence comparison and phylogenetic analysis of SWDs and crustins in penaeid shrimp suggest that the whey acidic protein domain(WAP) containing peptides/proteins can be divided into three classes:crustinâ… ,â…¡and SWD,and Fc-SWD is a new member in the SWD subgroup.Northern blot analysis demonstrated that the Fc-SWD transcripts were constitutively expressed in haemocytes of non-infected shrimp and increased in haemocytes of infected shrimp,but Fc-SWD mRNA was not detected in the hearts,hepatopancreas,stomachs,gills,intestines or ovarys of non-infected or infected shrimp.Reverse transcription polymerase chain reaction(RT-PCR) experiments showed that the Fc-SWD signal was detected in the haemocytes,hearts and gills of both unchallenged and challenged shrimp,and in stomach of challenged shrimp.In situ hybridization demonstrated that three cell types - hyaline,semi-granular, and granukar cell- in shrimp expressed Fc-SWD but only one of them has the strongest signals of expression.The cellular localization of the Fc-SWD protein in the haemocytes of the normal and the challenged shrimps was detected by immunocytochemistry.The peptide signal was predominantly located in the cytoplasm of some haemocytes,so the expression signal of Dc-SWD is probably in the cytoplasm of granular cells.Western blot analysis indicated that Fc-SWD protein was expressed in haemocytes and challenged haemolymphs,what's more,the molecular weight of the band found in haemocute samples was larger than that detected in the challenged haemolymphs.So the Fc-SWD protein appeared to be synthesized in haemocytes with a signal peptide and then secreted to the haemolymph as a mature peptide when F. chinensis was infected by pathogens.Fc-SWD(for the full length peptide including the signal peptide),mFc-SWD(for the mature peptide) and Fc-WAPD(for the single WAP domain) were expressed in Escherichia coli,respectively.Purified recombinant Fc-SWD and mFc-SWD have antimicrobial activities against gram-positive(G⁺) and gram-negative(G^-) bacteria, and fungi,as well as strong inhibitory activity against subtilisin A and protein K.The Fc-WAPD has anti-protease activity and low antimicrobial activity.These results suggest that Fc-SWD may be one of the key effectors in the shrimp immunity against various pathogens.Fc-SWD has antimicrobial and proteinase inhibition activities,and is a double function of antimicrobial peptides and broad prospects of bioactive peptides for development and medicinal value.2.Cloning and functional analysis of a glutamine synthetase in Chinese shrimp Fenneropenaeus chinensisGlutamine synthetase is a widely present in all organisms and distributed to 3 diffeent forms protein:GSâ… -family,mainly found in prokaryotes;GSâ…¡-family, mainly in eukaryotic Biology and in a small number of bacteria;GSâ…¢-family found mainly in prokaryotes.GS plays an important role in the immune system,nervous system,cell differentiation and proliferation,embryonic development,the synthesis of molecules, peritrophic membrane to form the matrix,and so on.A glutamine synthetase cDNA(Fc-GS) was cloned from Chinese shrimp Fenneropenaeus chinensis.The full length cDNA of Fc-GS is 1376 bp with an open reading frame(ORF) of 1086 bp encoding a protein of 361 residues.Fc-GS has a calculated molecular mass of 40.47 kDa and pI of 6.32.Alignment of amino acid sequences and phylogenetic analysis indicated that Fc-GS belongs to GSâ…¡-family.Expression of Fc-GS in haemocytes,heart,hepatopancreas,stomach,gill,intestine and ovary after bacterial- or white spot syndrome virus(WSSV)-challenged shrimps, and the time course expression of Fc-GS in hepatopancreas,gills and ovary of shrimps were investigated by RT-PCR.Our results showed that the Fe-GS was highly regulated in pathogen infected shrimp comparing to for all treatment.The Fc-GS mRNA level and protein concentration increased in the stomach and intestine in both bacterial and WSSV challenged shrimp.Expression of Fc-GS mRNA in haemocytes, gill,stomach and ovary was up-regulated after shrimp were injected with WSSV, while Fc-GS transcript was induced only in gill and stomach after bacterial infection.Protein expression of Fc-GS in hepatopancreas,heart,stomach,gill,ovary and muscles of the nomal shrimp,bacterial or white spot syndrome virus --challenged shrimps were analyzed by Western blot.The results indicated that Fc-GS was expressed in all tissues and increased in gill,stomach and ovary of bacteria-or WSSV-challenged shrimp.Fc-GS protein was also induced in the gill,stomach, muscle and intestine after bacterial or viral infection.Recombinant Fc-GS was expressed in Escherichia coli.The optimum pH for the recombinant Fc-GS(rFc-GS) was 6-7.5 and the optimum temperature was 37℃.The Fc-GS activity in muscles was increased significantly after bacterial or viral infection and the activity of Fc-GS enzyme in muscles was increased 2 folds in the shrimp infected with bacteria or WSSV compared to the uninfected shrimp.These results suggest that Fc-GS may play roles in immune responses against pathogen infection,especially viral infection.The Up-regulation of the gene and protein may be a biomarker for viral infection.The gene research provides a reference to understanding the Chinese shrimp glutamine synthetase involved in the immune-related signaling mechanism,and also provides a method basis to understanding the health state of Chinese shrimp after infected with bacteria or virus.3.Cloning and Characterization of Ras from the Chinese shrimp Fenneropenaeus chinensisProto-oncogene Ras is a multifunctional cytokine,the gene encoding the protein is an important signal transduction organizers,the cells directly involved in the pathway and other effects of cell signaling pathway.Ras protein is a small GTP-binding protein,the cell's outer membrane transfer of information plays an important role in cell signaling network known as the transmission of the "molecular switch."The full length of the cDNA of Fc-Ras of F.chinensis was 1013 bp including a 561 bp open reading frame that encoding 186 amino acids.There are three EGF₁ (EGF-like domain signature 1)(53-64,255-266,394-405),and one 2FE2S _FER₁(2Fe-2S ferredoxins,iron-sulfur binding region signature 1)(73-81) in open reading frame.Fc-Ras has a calculated molecular mass of 21 kDa and pI of 6.37.Haemocytes,heart,hepatopancreas,stomach,gills,intestine,ovary and spermary of normal and bacteria or WSSV challenged F.chinensis were selected to study the expression patterns of Fc-Ras with RT-PCR method.The data indicated the Fc-Ras gene expressed in all the selected tissues at different levels.There is a strong increased signal in Haemocytes,hepatopancreas,stomach,gills,and lower increase in intestine;but decreased in heart and ovary in bacteria-challenged shrimp.In the WSSV-challenged shrimp,there is a strong increased signal in Haemocytes,heart, stomach,gills,intestine,ovary and spermary,but no obviously increase in hepatopancreas.The results of western blot indicated that the Fc-Ras protein level was increased in stomach,intestine and ovary of bacteria-chanllenged and WSSV-challenged shrimp. Immunocytochemistry analysis of Fc-Ras in haemocytes indicated that the Fc-Ras strongly expressed in one type of shrimp haemocytes. It is of great significance to understand the role of innate immune-related signaling pathway when shrimp were in invasion by pathogenic microorganisms.In summary,a single whey acidic protein domain(SWD)-containing peptide has antimicrobial and proteinase inhibition activities.It is an important antimicrobial peptides for Fenneropenaeus chinensis innate immune defense.Fc-GS may be involved in the resistance of pathogenic micro-organisms,especially the resistance of virus.Fc-Ras may participate in the antiviral immunity and may be related to the signal transduction of Fenneropenaeus chinensis innate immunity.