Identification Of LGP2 And STING Genes In Yellow River Cyprinus Carpio And Their Genetic Polymorphisms Associated With Koi Herpesvirus Infection

Cyprinus carpio is one of the most important aquaculture fish in the world.However,in recent years,the frequent occurrence of various diseases of carp,so that the risk of carp breeding and economic losses are serious,but also seriously constrain the sustainable and healthy development of the industry.The acute gill necrosis disease is caused by Koi herpesvirus(KHV)has become the main hazard of carp breeding industry.The main symptoms are gill necrosis,eye depression,rough skin and other symptoms after KHV virus infection.KHV infection was first reported in the late 1990 s,until now,there are still not good defensive measures and healing means.A better understanding of the defense of C.carpio against this virus is essential to its prevention and the maintenance of a healthy aquaculture industry.The innate immune recognition of receptors and related genes from signaling pathways play crucial role in the virus detection,interferon induction and protection of the body.LGP2 is a member of the RIG-I-like pattern recognition receptor family that activates a series of signal cascades that recognize viral RNA and DNA in the cytoplasm to induce the production of type I interferon and proinflammatory cytokines.Interferon stimulating gene STING is a sensor for DNA virus recognition in host cells and is induced by interferon-mediated RIG-TBK1-IRF3/IRF7 signaling to induce innate immune responses to DNA pathogens.In order to study the molecular characteristics and antiviral mechanisms of some immune-related genes,the expression profiles of LGP2 and STING genes in vitro and in vivo were identified and analyzed by gene cloning,KHV virus infection and Poly(I: C)and poly(dA: dT)challenge,to explore the role of LGP2 and STING in virus recognition and antiviral cascade.In order to further explore the association and genetic characteristics of LGP2 and STING genes with anti-KHV,we assayed the DNA of the spleen of carp infected with KHV virus,and obtained the full length of LGP2 and STING genes by specific PCR and genome walking technique.And the bioinformatics and biostatistics related software tools were used to analyze the genetic information of the target gene.The promoter activity was verified by constructing an expression vector.The nucleotide polymorphisms of the target gene were analyzed by gene pool sequencing and PCR-RFLP,and the association with the phenotype was verified.1.There are two alternative splicing variants in the LGP2 gene of the common carp.The full-length cDNA of LGP2 a was identified as a 3,061-nucleotide cDNA sequence with an open reading frame encoding a putative protein of 680 aa,a 5?-untranslated region(UTR)of 126 nucleotides,and a 3?-UTR of 895 nucleotides,including a poly(A)tail.Analysis of conserved domains revealed the presence of a DEAD /DEAH box helicase(DExD/H)domain,a helicase super family C-terminal(HELICc)domain,and a regulatory domain(RD)at the C-terminus.The full-length cDNA of LGP2 b was identified as a 1,928-nucleotide cDNA sequence with an open reading frame encoding a putative protein of 346 aa,a 5?-untranslated region(UTR)of 114 nucleotides,and a 3?-UTR of 603 nucleotides,including a poly(A)tail.Analysis of conserved domains revealed the presence of a DEAD /DEAH box helicase(DExD/H)domain,3'UTR has 398 nucleotides with AATAA termination signal.LGP2 DNA of common carp was 7655 bp,with 12 exons and 11 introns.The splicing of introns is according with the ‘GT-AG' principle.The results showed that LGP2 a and LGP2 b derived from LGP2 DNA sequence by sequence alignment.However LGP2 a and LGP2 b appear different in the seventh exon at the mRNA sequence because the seventh intron part of the sequence is transcribed as a seventh exon and form a complete m RNA sequence.The 5 ‘flanking sequence of LGP2 is predicted with two promoters,and its activity can be enhanced by KHV induction.The results show that LGP2 plays an unanticipated role in the positive regulation of downstream responses both in vitro and in vivo as a component of innate cellular responses mediated by DNA pathogens.There are two possible mechanisms whereby LGP2 up-regulation could be involved in the anti-viral innate immune response: LGP2 could work upstream of RIG-I and MDA5 to potentiate viral-DNA-induced signaling,or LGP2 may be a node or branch point for coordinating crosstalk among seemingly diverse innate responses that are activated in infected cells.2.The 16 effective polymorphic loci of LGP2 were detected: including 12 SNP loci and 4 loci with deleted nucleotides;the-294 locus GCT trinucleotide deleted and 414 GT dinucleotide deleted site from the 5'flanking region.The genotype and allele frequencies of the 3280 T/C and 3836T/G in introns the nucleotide deleted sites were significantly different between the resistant and susceptible groups.The results showed that the carp mortality of-294 ins,414 ins,3820 CC and 3836 GG genotypes was lower than that of-294 del,414 del,3820TT and 3836 TT,respectively.Therefore,mutations in the four loci may be a genetic risk factor associated with koi herpes virus disease.3.The full length STING cDNA sequence contains 1528 bp,comprising an ORF of 1208 bp flanked by a 77 bp 5? UTR and a 243 bp 3? UTR with a canonical polyadenylation signal and a poly(A)tail.The cDNA encodes a polypeptide of 402 amino acids with a calculated molecular mass of 46.184 k Da and an isoelectronic point of 6.08.The deduced protein of STING contains a signal peptide and three transmembrane motifs(TM)in the N-terminal region.Four putative motifs(RXR)found in resident ER proteins are present in the C.carpio STING sequence.The full length of STING gene of C.carpio is 8068 bp,containing 7 exons,6 introns and a 5'-flanking region with 901 nucleotides;and the splicing of introns according with the ‘GT-AG' principle.The 5'-flanking sequence is 901 bp,the sequence is predicted with promoter activity,and its activity can be enhanced by KHV induction.The result shown that STING was an integral part of the synergistic response of the carp mediated by DNA pathogens,and confirmed plays a key role in vitro and in vivo for the forward regulation of downstream responses.4.The 14 valid polymorphic loci of STING were detected: 13 SNP loci,1 locus with deleted nucleotides;the-873G/C and-687 CCT trinucleotide deletion positions in the 5'flanking region the genotype and allele frequencies of the 6767G/A and 7034C/T loci in the exons were significantly different between the resistant and susceptible groups.Secondary virus stimulation experiments showed that the-873 GG,-687 ins,6767GG and 7034 CC genotypes of the STING gene had lower mortality rates than those of-873 CC,-687 del,6767TT and 7034TT(P <0.05),which were significantly associated with resistance/susceptibility to KHV infection and could be used as molecular markers for disease resistance breeding.5.In order to study the resistance mechanism of KHV virus and the association analysis of innate immune gene polymorphism with anti-KHV,the RNA was extracted from the gill and spleen in the infected carp with KHV for the transcripts sequenced.A total of 190 million raw reads and 135 million clean reads were obtained after filtering.469,251 transcripts and 366,783 unigene were assembled with the Trinity software.The average length of unigene was 667 bp,the maxmara was 18437 bp and the minimal was 201 bp.The length of 200-600 bp in the sequence of 347659 accounted for 74.08%.There were 31,837 sequences in the Nr database and had the highest coherence with zebrafish,followed by Mexican liqueur and rainbow trout,indicating the highest coherence with the Cyprinidae.The differentially expressed genes were screened by R with edgeR package,and the differences were analyzed by GO enrichment and KEGG pathway enrichment.The results shown that the differences in the resistance genes between the spleen and gills were similar to those of the susceptible group.The difference in expression between the 78 genes in the resistant and susceptible groups showed that the response of the fish to the anti-KHV virus in the spleen and gills was different.In this study,we also analyzed SNP,SSR and selective shear in the transcriptome library.The SNPs in the coding region were less than that in the non-coding region,but the coding region in the spleen was more than that in the non-coding region.The intentional mutation was 43.1%-50.58% of the total SNP,while the unintentional mutation only 0.11%-0.13%.In the library,the SNP converted from C to T is the largest,followed by T to C,and least is converted from C to G.In this database,31,084 SSRs were detected from the detection sequences 168,510,and the number of SSR-containing sequences was 25,240,accounting for about 15% of the total number of detected sequences,and the sequence containing more than one SSR was 4,549 18% of SSR sequences;1,401 for different base SSRs,and about 30.8% for more than one SSR.Repeat the maximum and minimum lengths of were 119 and 18,and an average length of 24 nucleotides.SSR of the type of sequence,mainly to single nucleotide 9-12 repeat were most,and 4 nucleotides and 5 nucleotides 5-8 repeat were the least.In this study,16,491 unigenes contained multiple transcripts(29.88%)by transcripts and unigenes,which may be regulated by selective shear.In these unigenes,called dierentially-expressed-transcript-containing genes(DET),which accounts for 7.58% of total Unigene(4,183).The results of this study provide a brief overview of the translocation group of the gill and spleen of the susceptible group and the gill and spleen of the disease-resistant group,and the first assessment of the immune biology of the carp against koi herpes virus,but also for the follow-up resistance to molecular breeding laid the theoretical basis.