A Functional Study Of Three Members Of The Trims Family In The Japonica Shrimp During WSSV Infection

Macrobrachium nipponense,also known as green prawns and prawns,is a kind of high quality freshwater prawns,widely distributed in rivers,lakes,reservoirs and ponds in China.Due to its unique taste,full of umami,rich in high protein,with high nutritional value,low fat content,it is favored by consumers on the table.Therefore,in recent years,M.nipponense has become an important economic breed in China,especially in Jiangsu and Zhejiang provinces.With the large-scale rearing of M.nipponense in each farm,the individuals are vulnerable to the infection of pathogenic microorganisms,resulting in economic losses,especially the white spot syndrome virus(WSSV).As an invertebrate,M.nipponense has no adaptive immune system,so it is urgent to study the innate immunity of M.nipponense.In this study,the effects of three important immune factors,MnTrim-like,MnTrim23 and MnTrim32,on innate immunity of M.nipponense were studied,and the following results were obtained:1.A gene was cloned from M.nipponense and named as MnTrim-like,with a total length of 1455 bp,including 963 bp open reading frame,encoding 321 amino acids.The protein has RING domain and consists of 42 amino acids.The gene is mainly expressed in hepatopancreas,stomach and intestines,but is less expressed in blood cells,heart and gills.WSSV infection can regulate its expression in stomach and intestine,and there are different degrees of upregulation.After knockdown of this gene,prawn were infected with WSSV.It was found that the expression of some antimicrobial peptides was significantly reduced and inhibited,and the copy number of WSSV and the expression of WSSV membrane protein gene VP28 were significantly increased.After interference with three antimicrobial peptides(Cru1,Cru6 and ALF1),the copy number of WSSV and VP28 were also significantly increased.WSSV and MnTrim-like protein co-incubated and injected into prawn significantly reduced the copy number of WSSV and VP28.Therefore,it is speculated that inhibition of virus replication in prawn plays an important role in the anti-WSSV immune response process.2.A gene that is highly similar to Trim23 in M.nipponense(MnTrim23).,the full length of the c DNA is 2295 bp,and it contains 1713 bp open reading frame,which can encode 571 amino acids.MnTrim23 protein has three conserved domains(a RING finger domain,two B-Box domains and a winding coil domain)at the N-terminal and one ARF domain at the C-terminal.The expression of MnTrim23 in intestines and hepatopancreas was higher than that in other immune tissues.The expression level of MnTrim23 in gills,stomach and intestines was notably up-regulated occurred after WSSV injection.In addition,silencing MnTrim23 inhibited the replication of WSSV virus and the expression of VP28,suggesting that MnTrim23 plays a positive role in WSSV infection.Further studies showed that MnTrim23 negatively regulates the expression of antimicrobial peptides(AMPs)mediated by Relish transcription factors.Drop-down AMPs promoted VP28 expression and WSSV copy number replication.These results suggest that Trim23 promotes WSSV replication by inhibiting AMPs expression,which is regulated by host NF-?B signaling pathway.3.The total length of MnTrim32 gene was 1757 bp,including 1206 bp open reading frame,and encoded 402 amino acids.The protein sequence encoded has the RING domain,which is composed of 43 amino acids,and 22 amino acids constitute the Coiled coil region of the other domain.The amount of expression was higher in the stomach and intestines than in other tissues.After WSSV infection,the expression of MnTrim32 in hemocytes,gills,stomach and intestines of prawn was significantly upregulated.Silencing of MnTrim32 inhibited the transcription of MnIRF,MnVago4 and MnTNF after WSSV infection,and significantly increased the expression of VP28 and the copy number of WSSV.After knockdown of MnIRF,MnVago4 and MnTNF,the expression of VP28 was detected and the copy number of WSSV was increased.Based on the results,we hypothesized that MnTrim32 is involved in antiviral innate immune response by regulating the expression of MnIRF,MnVago4 and MnTNF in M.nipponense.In this thesis,three genes with different domains in Trim family were studied,namely,MnTrim-like with RING domain only,MnTrim23 with typical domain,and MnTrim32 with RING domain plus a coiled helix region.The functions of three genes in response to WSSV infection were studied by using RNAi and qPCR techniques.