Molecular Cloning And Immune Response Analysis Of LGP2,MDA5 And MAVS Genes Of Bostrychus Sinensis

Bostrychus sinensis is a economic fish with high nutritional value in China,which is exported to Hong Kong and Macao.However,with the increase of the artificial aquaculture industry and the culture density,the number of various microorganisms has increased in the water environment.When microorganisms infect Bostrychus sinensis,it will induce related diseases and affect the development of the aquaculture industry,which is the main problem in the production process.Bostrychus sinensis is lower vertebrate,which is underdevelopment of the specific immunity,it rely on innate immunity to resist microbial invasion.The innate immune system mainly recognizes pathogen-associated molecular patterns(PAMPs)through three receptor families of TLRs,NLRs and RLRs to play an immune response,which induces the expression of pro-inflammatory cytokines and interferon.Thereinto,RLRs are a novel family of pattern recognition receptor for intracellular recognition of viruses,which play an important role in antiviral immune response.The RLRs family are consists of three members,LGP2(Laboratory of Genetics and Physiology 2),RIG-I(retinoic acid-inducible gene 1)and MDA5(melanoma differentiation-associated gene 5),respectively.Structurally,RIG-I and MDA5 protein have two CARD domains at N-terminal,which can interact with MAVS located on the mitochondrial membrane to activate immune pathway by CARD-CARD domain.Although LGP2 lacks CARD domain,it also has complex regulatory effects.In this study,according to the existing gene sequences in the transcriptome of the laboratory,the LGP2,MDA5 and MAVS genes of Bostrychus sinensis were cloned by PCR amplification,and the molecular characteristics were analyzed by bioinformatics methods.Real-time PCR was used to detect the expression level changes of LGP2,MDA5 and MAVS genes of Bostrychus sinensis after poly(I:C)infection to speculate the role of LGP2,MDA5 and MAVS genes in antiviral immunity of Bostrychus sinensis.The coding sequences of LGP2 gene(named BsLGP2)of Bostrychus sinensis is 2040 bp,encoding 679 amino acid residues.Protein domain prediction found that BsLGP2 includes DEXDc,HELICc and RD three conserved domain.The results of Real-time PCR showed that BsLGP2 gene was ubiquitously expressed in 10 tissues of healthy Bostrychus sinensis,with the highest expressions in the head kidney,but very low in the intestine.The expression of BsLGP2 gene was significantly up-regulated at the early stage of other immune organs except spleen after poly(I:C)injection.The expression level of BsLGP2 was continuously up-regulated in peripheral blood(48 h,about 158 times);the expression level of BsLGP2 gene peaked at 12 h in the head kidney and liver(about 17 times and about 70 times);while the expression of BsLGP2 gene was down-regulated at early stage in the spleen,the expression level was rapidly up-regulated after 12 h and reached the highest peak at 24 h(about 1.3 times).BsLGP2 gene may play a positive regulatory role in innate antiviral immunity.The coding sequences of BsMDA5 is 2988 bp,encoding 995 amino acid residues.It is predicted that BsMDA5 protein domains are conserved,they are CARDs,DEXDc,HELICc and RD,respectively.BsMDA5 gene was expressed in all the tested tissues,with the highest expression in skin,followed by peripheral blood,but very low in intestine and brain.The expression of BsMDA5 gene was rapidly up-regulated at the immune tissues after poly(I:C)stimulation compared with control group(PBS),and showed increased firstly to the peak and then declined rapidly.In peripheral blood,head kidney and liver,the expression level of BsMDA5 was significantly up-regulated at 3 h,and reached the highest expression level at 12 h(about 30 times,about 3.78 times,about 21.78 times).The expression of BsMDA5 gene in spleen reached peak at 24 h(about 3.22 times),indicating that MDA5 gene plays an important regulatory role in innate immune response.The coding sequences of BsMAVS is 2484 bp,encoding 827 amino acid residues.It is predicted that BsMAVS protein includes one conserved CARD domain and two internal repeats(RPT1).BsMAVS gene was widely expressed in noraml tissues by Real-time PCR,with the highest expressions in head kidney and spleen,but very low in intestine.In the peripheral blood,head kidney,liver and spleen stimulated by poly(I:C),the expression of BsMAVS gene in peripheral blood was rapidly and significantly up-regulated(48 h,about 30 times);in the head kidney,the expression of BsMAVS gene was down-regulated from 3 h to 12 h,the expression was significantly up-regulated after 12 h and reached the highest peak at 24 h(about 1.3 times);the expression of BsMAVS gene was up-regulated and reached peak at the early stage(3 h,about 1.53 times)in liver;the expression of BsMAVS gene was down-regulated at the early stage in the spleen,the expression was rapidly increased after 6 h and peaked at 12 h(about 1.2 times).It is speculated that BsMAVS gene plays an important role in innate immune response.In short,the study the LGP2,MDA5 and MAVS genes coding sequences of Bostrychus sinensis were cloned,and the expression of LGP2,MDA5 and MAVS genes were preliminarily studied.These results showed that BsLGP2 may play a positive regulatory role in innate antiviral immunity and is a positive regulatory factor in RLR antiviral signaling pathway;BsMDA5 gene may be involved in the innate antiviral immune response and plays an important role;although the BsMAVS gene lacks TM domain,it still plays a role in innate antiviral immune response.It lays a foundation for the study of RLR signaling pathway and related molecular mechanism of Bostrychus sinensis,and playing a guiding role in the prevention and control of Bostrychus sinensis disease.