| Blood clam(Tegillarca granosa)is a kind of marine bivalve shellfish with high economic value.In recent years,the production of blood clam has been seriously restricted due to the disease caused by marine Vibrio.Therefore,by means of molecular biology,the culture process of blood clam infected by natural Vibrio was simulated in order to deeply study the signal transduction of immune pathway in blood clams and analyze the defense mechanism of innate immune system of blood clam.It can provide a basic molecular reference for the cultivation of blood clam resistant to Vibrio infection.Activating protein-1(AP-1)is an important nuclear transcription factor.Through the basic leucine zipper structure(b ZIP)of its dimer,AP-1 can directly regulate gene transcription containing TPA response elements(TRE)to respond to a variety of environmental stress stimuli.It plays an important role in the innate immunity of marine bivalves against virus or bacterial infection.In this study,through the common annotation of genome and transcriptome of blood clam,the AP-1homologous gene in blood clam was identified.The response mechanism of AP-1immune-related pathway including upstream and downstream related genes in innate immune defense of blood clam was analyzed.In addition,the molecular markers and haplotypes related to AP-1 resistance were found.The main results of the study are as follows:1.The AP-1 homologous gene(TgAP-1)was cloned from blood clam.The total length of the c DNA sequence of TgAP-1 is 1591 bp,with an ORF of 879 bp,encoding 292 amino acids,and the protein molecular weight is 32.8 k Da.TgAP-1protein contains an N-terminal Jun domain and a conserved basic leucine zipper domain(b ZIP).Multiple sequence alignment and phylogenetic tree analysis showed that TgAP-1 was relatively conserved and belonged to invertebrate bivalve clade,with the highest homology with Mytilus edulis.Relative fluorescence quantification(RTPCR)and protein western blotting(WB)showed that TgAP-1 was generally expressed in blood clam tissues,among which the expression level was highest in gill.Immersion challenge by Vibrio harveyi could significantly up-regulate the expression level of TgAP-1 in the gill tissues of blood clam(P < 0.05).Especially within 24 h after immersion,the expression level of TgAP-1 in the gill tissues of the blood clam continued to increase.It suggested that TgAP-1 was involved in the immune response to V.harveyi infection of the blood clam.2.c-Jun N-terminal kinase(JNK),a potential regulatory gene upstream of AP-1 was located by reference signal pathway.The c DNA sequence of JNK(TgJNK)was obtained by cloning in blood clam.The regulatory relationship of TgJNK and TgAP-1 under V.harveyi infection were analyzed.The results showed that the full length of TgJNK c DNA is 2696 bp with an ORF of 1332 bp,and 443 amino acids are encoded.The protein molecular weight is 50.6 k Da.Multiple protein comparisons and phylogenetic tree analysis showed that TgJNK was relatively conserved,with a STKc-JNK domain,and has high homology similarity with Crassostrea gigas.The results of RT-PCR and WB showed that TgJNK was the highest expression in the gill tissue of blood clam.V.harveyi immersion infection significantly upregulated TgJNK transcription levels(P < 0.05)and phosphorylation levels,and increased the protein abundance of TgAP-1.The transcription levels of TgJNK and TgAP-1 were significantly decreased(P < 0.05),and the phosphorylation levels of TgJNK and the abundance of TgAP-1 protein were also decreased after JNK inhibitor treatment.Protein co-immunoprecipitation(Co IP)analysis showed that the phosphorylated JNK protein had a binding relationship with TgAP-1 protein.Studies have shown that TgJNK and TgAP-1 are involved in the innate immune response of blood clam after infection with V.harveyi.Phosphorylation of TgJNK protein was an important factor in activating TgAP-1 protein,and TgJNK could positively regulate the expression of TgAP-1.3.Based on RNAi detection,the Tg IL-17 D of inflammatory cytokine IL-17family(IL-17),the target gene of AP-1 pathway,was successfully cloned and sequenced.It was proved to be involved in immune and inflammatory response after V.harveyi infection.Tg IL-17 D c DNA is 1587 bp with an ORF of 594 bp,and encodes197 amino acids.The protein molecular weight is 22.0 k Da.RT-PCR and WB detection showed that Tg IL-17 D was expressed in all tested blood clam tissues,and the expression level of gill was the highest.Gel electrophoresis migration(EMSA)and chromatin immunoprecipitation(Ch IP)analysis showed that there were multiple TgAP-1 protein binding sites in the Tg IL-17 D promoter.The inflammatory reaction,physiological metabolism and death of blood clam after V.harveyi infection were analyzed by knocking down TgAP-1 gene.The results showed that V.harveyi stress could significantly up-regulate the expression of TgAP-1 and Tg IL-17D(P < 0.05),and down-regulate the expression of TgAP-1 could significantly decrease the expression of Tg IL-17 D in gill tissue of blood clam(P < 0.05).In addition,the deficiency in the expression of TgAP-1 reduced the pro-inflammatory response caused by inflammatory cytokine Tg IL-17 D after V.harveyi infection,and reduced the apoptosis and necrosis of tissue cells,not only maintained the normalization of physiological metabolism,but also prevented the large-scale stress death of infected blood clam.4.By the third generation of molecular markers Single nucleotide polymorphism(SNP)to find a potential site which TgAP-1 related to resistance of V.harveyi.A total of 18 SNPs sites were detected in the blood clam population by highthroughput sequencing.According to the SNPs typing of resistant blood clam population 09-R,control blood clam population 09-C,resistant blood clam family 17-R and susceptible blood clam family 17-S and the analysis of linkage disequilibrium,Six SNPs(AG type of Tg SNP-1,GA type of Tg SNP-2,TG type of Tg SNP-4,CT type of Tg SNP-7,AG type of Tg SNP-11,and G A type of Tg SNP-12)and four haplotypes(f Hap2,f Hap3,f Hap6,and f Hap7)were significantly associated with resistance to V.harveyi.Risk assessment showed that f Hap2(CG)and f Hap7(GA)were associated with an increased resistance,while f Hap3(CT)and f Hap6(AG)were associated with an increased susceptibility.In addition,the expression level of TgAP-1 in the tissue of resistant blood clam population 09-R was significantly lower than that of control blood clam population 09-C(P<0.05).After V.harveyi infection,the expression level of TgAP-1 in the gill tissue of resistant blood clam population 09-R was still significantly lower than that of control blood clam population 09-C(P<0.05).It was speculated that the difference in the expression of the TgAP-1 gene in different populations with resistance to blood clam was an important factor for the difference in the trait of resistance to V.harveyi blood clam,and such resistance trait difference was likely to be affected by the genotype difference caused by the single nucleotide polymorphism of the TgAP-1 gene.This study proved that AP-1-centered signal pathway genes TgJNK,TgAP-1and Tg IL-17 D were involved in the innate immune response of blood clam infected with V.harveyi.Phosphorylation of TgJNK protein was an important factor in activating TgAP-1 protein,and TgJNK could positively regulate the expression of TgAP-1.TgAP-1 played an important role in the regulation of Tg IL-17 D expression.By knocking down TgAP-1 and down-regulating the expression of Tg IL-17 D,it could protect blood clam from inflammatory damage after V.harveyi infection and reduce stress death caused by V.harveyi infection.At the same time,in different resistant materials,it was also found that the low expression of TgAP-1 was an important factor in the formation of V.harveyi resistance of blood clams.In addition,the identification of genetic molecular markers and haplotypes related to V.harveyi resistance not only enriches the theoretical knowledge of innate immunity of bivalve shellfish such as blood clam,but also provides scientific guidance for molecularassisted breeding of bivalve shellfish such as blood clam in the future. |
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