| Macrobrachium nipponense,which is widely distributed in various regions of China,is a very important freshwater cultured shrimp.However,in recent years,high mortality and huge economic losses have been brought to shrimp culture due to the abuse of bacteria,fungi,viruses,pesticides and antibiotics.How to effectively prevent the occurrence of shrimp diseases is extremely important for aquaculture,and the study of shrimp innate immune defense mechanism can effectively guide disease prevention and control in production practice.Shrimp are lack of acquired immunity,which mainly rely on innate immunity to resist the invasion of various pathogens.Pattern recognition receptors and immune signal pathways,as the key components of innate immune system,play an important role in the process of invertebrate fighting against pathogenic microorganism infection.In this article,we studied the innate immune defense function of three important immune genes AP-1(activator protein 1),ATF2(Activating transcription factor 2)and Fos of AP-1 transcription factor family in M.nipponense,and obtained the following results.(1)Study on innate immune function of MnAP-1 gene in M.nipponenseTranscription factor AP-1 plays an irreplaceable role in cell stress,bacterial and viral infection,inflammatory cytokines and other external stimuli.In this study,a new AP-1 gene,named MnAP-1,was cloned from M.nipponense.The full length of the gene is 1747 bp,which contains the open reading frame of 882 bp and encodes a protein of 293 amino acids.MnAP-1 protein contains Pfam and b ZIP domains.MnAP-1 is widely distributed in hemocytes,heart,hepatopancreas,gill,stomach and intestinal tissues.After the challenge of Vibrio parahaemolyticus and Staphylococcus aureus the expression of MnAP-1 in gill and stomach was significantly up-regulated.We used RNA interference to study the relationship between MnAP-1 and(AMPs)transcripts of antimicrobial peptides in gill.Interestingly,MnAP-1 has different regulatory effects on the expression of different AMPs.We found that the expression of Cru1,Cru3 and Cru4 in gill was significantly decreased,while the synthesis of Cru5 and anti-lipopolysaccharide factors(ALF3 and ALF4)was significantly increased.We further discussed the effect of MnAP-1 on the expression of transcription factor Relish in M.nipponense.The results showed that MnAP-1 gene knockdown could significantly up-regulate the expression of MnRelish.As a member of the NF-?B family,it regulates the expression of AMPs in the innate immunity of crustaceans.Therefore,we also detected the expression levels of Cru5,ALF3 and ALF4 in the gills of M.nipponense silenced by MnRelish.The data showed that the expression levels of these three kinds of AMPs decreased significantly after MnRelish silencing.The results showed that MnAP-1 positively regulated the expression of AMPs,promoted JNK / AP-1 signal pathway and negatively regulated the synthesis of AMPs by inhibiting the transcription of NF-?B factor Relish in innate immunity of M.nipponense.(2)Cloning and identification of MnATF2 gene in M.nipponenseTranscription factor ATF2 is a member of the b ZIP transcription factor family,which participates in a variety of physiological and developmental processes,but its role in innate immunity is not clear.In this study,two subtypes of ATF2 gene(MnATF2 a and MnATF2b)of M.nipponense were identified,which were produced by exon skipping.The full-length MnATF2 a is 2328 bp,with an open reading frame of 2079 bp,encoding 692 amino acids.MnATF2 a has more 237 bp nucleotides than MnATF2 b,and the extra 237 bp is a complete exon.MnATF2 a and MnATF2 b proteins have the same conserved and typical b ZIP domains at the C-terminal.MnATF2 a has 79 more amino acids than MnATF2 b.MnATF2 a and MnATF2 b are widely distributed in a variety of immune tissues.12 hours after V.parahaemolyticus and S.aureus infection,the expression of MnATF2 a and MnATF2 b in gill and stomach was significantly up-regulated.RNA interference analysis showed that MnATF2 gene knockdown significantly inhibited the transcription of tumor necrosis factor(TNF)and promoted the expression of Cru3,Cru4 and Cru7.Further studies showed that knockdown of MnTNF gene significantly increased the expression of Cru3,Cru4 and Cru7.Our study shows that ATF2 negatively regulates the expression of AMPs by regulating the transcription of TNF in M.nipponense.This study provides valuable information for the study of the role of ATF2 family in crustacean innate immunity.(3)Study on the innate immune mechanism of MnFos gene in M.nipponenseIn this paper,we first reported the isolation and identification of the full-length sequence of Fos gene(named MnFos)in M.nipponense,and found that six isomers of MnFos are produced by 5' alternative first exon and exon skipping.The full-length amino acid sequence of MnFos protein contains a basic leucine zipper domain(b ZIP),which is the main characteristic of AP-1 family members.Through the tissue distribution experiment,the results showed that MnFos gene existed in all tissues of M.nipponense.After white spot syndrome virus infection,the gill,stomach and intestine of M.nipponense were detected by q RT-PCR.The results showed that MnFos gene was significantly up-regulated in gill,stomach and intestine during virus infection,which indicated that MnFos played a crucial role in the innate immunity of M.nipponense.According to the related literature,it has been reported that Fos gene can activate various transcription factors and crosstalk signal pathways in shrimp innate immune defense,especially in antivirus.Using RNA interference(RNAi)to inhibit the transcriptional level of MnFos and detect the expression of related antimicrobial peptides,it was found that the expression of Cru1,Cru5,Cru6 was significantly increased;silencing MnFos gene under WSSV infection,and detecting the expression of Cru1,Cru5,Cru6,it was found that the expression level was also significantly increased,indicating that MnFos may be involved in the mechanism of WSSV replication and pathogenesis.Subsequently,the transcription of viral envelope protein VP28 was detected,and the expression of VP28 was significantly down-regulated.The results showed that the knockdown of Fos gene would affect the transcriptional level of VP28.The experimental results in this chapter show that the MnFos gene helps to replicate in the virus host by inhibiting the expression of downstream related antimicrobial peptides.In a word,the experimental results of this paper provide strong evidence and novel insights for the study of the role of MnFos in virus infection.In summary,members of the AP-1 family participate in host immunity in different ways,which provides a reliable theoretical support for disease prevention and control of M.nipponense. |
PDF Full Text Request