The Function Study Of Three Immune-related Genes Caspase-3C, Lectin And LGBP In Procambarus Clarkii

Procambarus clarkii,commonly known as small lobster or red crayfish,are omnivorous,fast-growing and easily adaptable.It was enjoyed by people for its delicious since being introduced into our country in 1930s,and then occupied the important position of freshwater shrimp economic industry.It is now one of the most valuable shrimp products in the world.More than ten years ago,the"lobster gate"event in nanjing made people pay more attention to the disease of crayfish.In addition,as the passion for crayfish continues to grow,demand exceeds supply,for which aquaculture farmers breed crayfish on a larger scale.Consequently,crayfish are more likely to get sick and the pathogens become more diverse.However,like other invertebrates,the crayfish only rely on innate immunity to defend against pathogen invasion.Therefore,the study of innate immunity is particularly important for crayfish.In this study,we studied the immune functions of Caspase-3C,Lectin and LGBP in P.clarkii,and obtained the following results:1.A new cDNA named PcCaspase-3C was found in P.clarkii with a full-length of4684 bp,and a 1572 bp opening reading frame?ORF?is included,which encodes a putative protein of 523 amino acids.PcCaspase-3C also contains a CASc domain with237 amino acids and a low complexity region.The PcCaspase-3C was mainly expressed in heart,stomach and intestine,and less in the hepatopancreas and gills,with least expression in haemocytes.Had been infected with Staphyloccocus aureus,Vibrio parahaemolyticus or white spot syndrome virus?WSSV?,there was an up-regulated expression level of PcCaspase-3C in intestine or stomach to varying degrees.When crayfish were infected with V.parahaemolyticus after the RNA interference of PcCaspase-3C,some antimicrobial peptides such as ALF2,ALF5,ALF6,Cru4 or Lys were inhibited.In addition,when crayfish were infected with WSSV after the RNA interference of PcCaspase-3C,the WSSV copies were increased significantly.According to these results,we suggest that the PcCaspase-3C may play a crucial role in the antibacterial and antiviral immune responses of P.clarkii.2.C-type lectins,as one kind of pattern recognition receptors?PRRs?,play important roles in innate immune system of crustaceans.In this study,a new cDNA named PcLectin was found in P.clarkii with a full-length of 1025 bp,and a 528 bp opening reading frame is included,which encodes a putative protein of 175 amino acids.PcLectin also contained a carbohydrate-recognition domain?CRD?with 145 amino acids,and a signal peptide with 23 amino acids.The PcLectin was enriched expressed in stomach,less in hepatopancreas,and rarely in other tissues.The various upregulation of PcLectin was manifested in crayfish challenged with S.aureus,V.parahaemolyticus or WSSV.After the RNA interference of PcLectin,V.parahaemolyticus was used to stimulate the crayfish,and the expression of some antimicrobial peptides were inhibited?ALF2,ALF6,Cru2,Cru4?.The gene was recombinant expressed in vitro,and the recombinant protein PcLectin?rPcLectin?had binding activity towards Gram-positive,Gram-negative bacteria and polysaccharides.The rPcLectin could also agglutinate bacteria in a Ca²⁺-dependent manner.According to these results,we suggest that the PcLectin might be involved in the innate immune defense of P.clarkii.3.As a pattern recognition receptor,the lipopolysaccharide and?-1,3-glucan binding protein?LGBP?participates in the innate immunity of invertebrates.In this study,a new gene named PcLGBP was cloned from P.clarkii with a full length of 1327bp,and it included an ORF of 1113 bp encoding a protein composed of 370 amino acid residues.PcLGBP also contained a signal peptide,a glycosidase activity domain?Glyco_hydro₁6?and a low-complexity region.It was found that there were gender differences in the expression of this gene in different tissues.In healthy crayfish,the PcLGBP in females was almost lower than that in males.Both female and male crayfish had the highest expression of PcLGBP in hepatopancreas,less in other tissues with least in the intestine.After the stimulation of V.parahaemolyticus or lipopolysaccharides?LPS?,the changes of the gene in females and males were also different,but the expression of the gene in the stomach and hepatopanpancreas showed an overall upward trend.In hepatopanpancreas of males,PcLGBP was successfully interfered,and the expression of multiple antimicrobial peptides was inhibited.The recombinant protein LGBP?rPcLGBP?was determined to have binding activity to LPS,Glu and PGN in different degree,and could bind a variety of Gram-positive and Gram-negative bacteria.For phenoloxidase?PO?activity,moreover,the experimental results showed that in normal hepatopanpancreas,gills and stomach of males,the PO activity was significantly higher than that of females.After the RNA interference of PcLGBP in males,the PO activity was significantly decreased,and there was an obviously increase after the rPcLGBP being incubated with ?-1,3-glucan?Glu?or LPS.The above results showed that LGBP both can participate in the regulation of antimicrobial peptides and the activation of phenol oxidase in P.clarkii.And the high expression of PcLGBP endowed males with high PO activity.