The Preliminary Functional Study Of Fibrinogen-related Proteins And Kruppel-like Factors In The Innate Immunity Of Macrobrachium Rosenbergii

It is generally believed that vertebrates have both innate immunity and acquired immunity.In invertebrates,because of lack of real antibodies and specific immune cells,the body's defense response is only innate immunity.In this paper,the innate immunity of Macrobrachium rosenbergii was studied.1.Transcriptome analysis of lymphoid organs of M.rosenbergii after WSSV stimulation.M.rosenbergii is a crustacean with economic importance,and adult prawns are generally thought to be tolerant to white spot syndrome virus(WSSV)infection.Although certain genes are known to respond to WSSV infection and lymphoid tissue is an important immune organ,the response of lymphoid organ to WSSV infection is unclear.Next-generation sequencing was employed in this study to determine the transcriptome differences between WSSV infection and mock lymphoid organs.A total of 44,606,694 and 40,384,856 clean reads were generated and assembled into 73,658 and 72,374 unigenes from the control sample and the WSSV infection sample,respectively.Based on homology searches,KEGG,GO,and COG analysis,21,323 unigenes were annotated.Among them,4951 differential expression genes were identified and categorized into 244 metabolic pathways.Coagulation cascades,and pattern recognition receptor signaling pathways were used as examples to discuss the response of host to WSSV infection.We also identified 12,308 simple sequence repeats,which can be further used as functional markers.Results contribute to a better understanding of the immune response of prawn lymphoid organ to WSSV and provide information for identifying novel genes in the absence of the prawn genome.2.Fibrinogen-like lectin,as a pattern recognition receptor,participates in the antibacterial innate immune response in M.rosenbergiiFibrinogen-like lectins are widely present in vertebrates and invertebrates.In this study,a fibrinogen-like lectin gene was cloned from M.rosenbergii and named as MrFREP.The full-length cDNA of MrFREP from M.rosenbergii is 1649 bp in length.It contains a 1086 bp open reading frame(ORF)encoding a protein with 361 amino acid residues,a 74 bp UTR at the 5' end.The 3' end contains a 492 bp UTR with a poly A tail.MrFREP contains a 20 amino acid signal peptide and a 223 amino acid carbohydrate recognition domain.The phylogenetic tree showed that the relationship between MrFREP and MjFREP was the closest,with 43%similarity.Through analysis of tissue distribution,it can be seen that MrFREP is expressed in the hemocytes,heart,hepatopancreas,gills,stomach,intestine and muscle of M.rosenbergii,and has a high expression level in gills and intestine.The expression level of MrFREP increased at 24 hours after stimulation with Vibrio parahaemolyticus and white spot syndrome virus(WSSV).MrFREP recombinant protein can agglutinate Gram-positive bacteria and Gram-negative bacteria in the presence of Ca2+ and can also bind with Gram-positive bacteria and Gram-negative bacteria.In addition,the recombinant protein can also bind lipopolysaccharide and peptidoglycan,and significantly improve the efficiency of Vibrio clearance in shrimp.3.Kruppel-like factors participate in the regulation of innate immune responses of M.rosenbergiiThe Kruppel-like factor family is widely present in eukaryotes.The full-length cDNA of Kruppel-like factor from M.rosenbergii(MrKLF)is 1799 bp.It contains a 1335 bp open reading frame(ORF),which encodes a protein with 444 amino acid residues.It also contains a 193 bp non-coding region at the 5' end and a 3'-end(274 bp long)contains a poly A tail.MrKru contains a 12-amino acid and a 13-amino acid low-complexity region and three zinc finger protein domains consisting of 25,25,and 23 amino acids,respectively.The phylogenetic tree indicated that Kruppel-like factor of M.rosenbergii had the closest genetic relationship with Kruppel-like factors in Penaeus monodon and Penaeus vannamei,and the similarities were 59%and 58%,respectively.At the mRNA level,the Kruppel-like factor gene is widely distributed in M.rosenbergii,and the expression level in hepatopancreas,gills,and intestine is significantly higher than that in other tissues.When stimulated by white spot syndrome virus and V.parahaemolyticus,the expression level of MrKLF firstly decreased significantly at 24 hours and increased at 48 hours.When the gene was knocked down,the expression levels of some antimicrobial peptide genes(AMPs)in prawns was inhibited.The results suggested that AMPs could be regulated by MrKLF.It could be included that MrKLF may be involved in the innate immune defense of M.rosenbergii.