Functional Study Of Bursicons, Relish And LGR2 In Innate Immunity Of Macrobrachium Japonicus

Macrobrachium nipponense,also known as river prawn,freshwater prawn,is widely distributed in some freshwater of China and other Asian countries.As one of the most important freshwater species in China,M.nipponense has strong adaptability and wide distribution,which occupies an important position in the freshwater economic crustacean culture industry.In recent years,due to the development of intensive farming,the disease of M.nipponense occurred frequently.As an invertebrate,M.nipponense lacks specific immunity and only has innate immune system to resist the disease.Therefore,it is very important to study the innate immune mechanism of M.nipponense.This study studied the immune function of Bursicon,Relish and LGR2,three important possibly immune-related genes,in M.nipponense,and obtained the following results:1.Bursicon is a neuropeptide hormone related to molting,which has been found to play an important role in the innate immunity of invertebrates.This paper focuses on the role of Bursicon gene in the innate immunity of M.nipponense.The full-length sequences of two subunits of Mn Burcison(Mn Bursicon-? and Mn Bursicon-?)were obtained by homologous cloning,respectively.In this study,we discussed the two subunits respectively.The total length of Mn Bursicon-? sequence was 959 bp,and the open reading frame was 444 bp,which contained 147 amino acids.The Mn Bursicon-?sequence is 1370 bp in length,with an open reading frame of 414 bp and contains 137 amino acids.Both Mn Bursicon-? and Mn Bursicon-? protein contain a signal peptide and a C-terminal cystine knot-like domain(CTCK).Mn Brsicon? is mainly expressed in hepatopancreas,hemocytes and intestines,but is low in heart,stomach and gills,while Mn Brsicon? is mainly expressed in hepatopancreas,stomach and intestines,but is low in gills,heart and hemocytes.The expression levels of Mn Brsicon? and Mn Brsicon? in gill and stomach tissues of prawn were upregulated by Staphyloccocus aureus,Vibrio parahaemolyticus or white spot syndrome virus(WSSV).Mn Bursicon-? and Mn Bursicon-? were knocked down by injection of double-stranded RNA,and the expressions of antimicrobial peptides were detected respectively.The expressions of six antimicrobial peptides genes were inhibited(Mn Cru1,2,3,Mn ALF1,2 and Mn Car1).At the same time,bovine serum protein,the recombinant proteins of Bursicon-? and Bursicon-? were injected respectively,and the expression of antimicrobial peptides was detected again.Compared with the control group,the expression levels of the same six antimicrobial peptides(Mn Cru1,2,3,Mn ALF1,2 and Mn Car1)were increased.Therefore,we believe that Mn Burcison is involved in the innate immune process of M.nipponense by regulating the expression of antimicrobial peptides.2.Relish is a nuclear transcription factor in the IMD signaling pathway,belonging to the NF-?B protein family,and plays an important role in the nuclear regulation of antimicrobial peptide expression.We cloned the full-length sequence of Mn Relish in M.nipponense,and found that the full-length sequence of Mn Relish was5082 bp,in which the open reading frame was 3549 bp long,encoding a peptide chain of 1182 amino acids.The protein consists of an N-terminal Rel homologous domain(RHD),an IPT domain,four low-density domains,six ankyrin repeats and one coiled coil region.The expression level of Mn Relish was highest in gill tissues,followed by hemocytes and hepatopancreas,and low in heart,stomach and intestine tissues.Under the infection of S.aureus,V.parahaemolyticus and WSSV,the expression levels of Mn Relish in gills and stomach tissues of prawn were upregulated to different degrees.After Mn Relish was knocked down,the expression of five antimicrobial peptides(Mn Cru1,Mn Cru2,Mn Cru6,Mn ALF1 and Mn ALF2)were down-regulated by the detection of antimicrobial peptide expression.At the same time,the Mn Relish gene was knocked down again,the bovine serum protein and the recombinant protein of Burcison?/? was injected respectively.The results showed that the expression of three antimicrobial peptides(Mn Cru1,Mn ALF1 and Mn ALF3)in the Burcison?/?recombinant protein group was significantly down-regulated after Mn Relish gene knockdown compared with that of bovine serum protein injection.Therefore,we believe that Mn Relish is an important transcription factor that regulates the expression of antimicrobial peptides and participates in the process of innate immunity.3.LGR is a G-protein-coupled receptor which cantains Leucine-rich repeats,belonging to a membrane binding receptor.We obtained a full-length sequence of LGR from M.nipponense by homologous cloning,which was identified as type B LGR by biological analysis and named as Mn LGR2.The length of Mn LGR2 sequence is 5921 bp,with an open reading frame of 4515 bp,encoding a peptide chain of 1504 amino acids.The protein contains a 7-transmembrane domain,12 leucine-rich repeats,and 5 low-complexity regions.The expression level of Mn LGR2 was the highest in gill tissue,but not high in stomach,heart,intestine,hepatopancreas and hemocytes.In the prawn infected with S.aureus,V.parahaemolyticus and WSSV,the expression level of Mn LGR2 all changed to different degrees.After knockdown of the gene,the expression of antimicrobial peptides was detected,and the expression levels of five antimicrobial peptides(Mn Cru1,Mn Cru2,Mn Cru6,Mn ALF1 and Mn ALF2)were found to be increased.Meanwhile,the expression level of Mn Relish was detected,and it was found that the expression level of Mn Relish was increased when the expression of Mn LGR2 was inhibited.After knockdown of Mn LGR2,we detected the expression levels of related genes in the pro PO pathway,and found that the expression levels of 4related genes were all down-regulated(Mn PPAE1,2,3 and Mn PPAF),and the PO activity also showed a down-regulated trend.In conclusion,we hypothesized that Mn LGR2 promotes the expression of genes related to the pro PO pathway and increases the activity of PO by inhibiting the expression of antimicrobial peptides.