Cloning, Expression And Linkage Mapping Of Four Complement Genes And Their Association With Resistance To Bacterial Hemorrhagic Disease In Grass Carp Ctenopharyngodon Idellus

Grass carp is one of the most impotant fresh aquaculture fishes in China. The yield andoutput value of grass carp are ranked first one of all aquaculture fishes in China and Inthe world. Grass carp is easy to be infected by many pathogens. Various diseases cancause heavy losses in grass carp aquaculture every year. So functional studies on itsimmune factors affecting resistance to diseases were carried out to understand theirrelationship with disease resistance, then will provide the theoretical base for molecularbreeding of grass carp. In this thesis, the grass carp complement Bf/C2, C6and C7genes were cloning and characterized, and the linkage and expression were analyzed tounderstand their biological function. The polymorphisms of complement C6and C7were investigated, and genetype and haplotype were associated with resistance tohemorrhagic disease in grass carp.1. Molecular cloning and expression of four complement gene in grass carpThe primers were designed according to the contigs or zebrafish cDNA sequences. Thepart of the full-length fragments of four grass carp complement genes were isolated, andthe four genes were Bf/C2A、Bf/C2B、C6、C7. RACE method was used to clone four genesin grass carp. The amino acid sequences of the four genes were deduced and comparedwith some of other species close genetically. The full-length cDNA sequences of thisfour genes were submitted to GenBank, and the accession numbers are JF47038,JF47039, HQ416903, JN655169.Real time PCR method was used to investigate the four grass carp genesexpression patterns in various tissues of healthy fish, at different developmental stages,after challenge with Aeromonas hydrophila bacterium. The results show that Bf/C2A,Bf/C2B, C6genes were expressed in a wide range of grass carp tissues, with the highestlevel of expression of these genes detected in the liver and spleen, but C7geneexpression was detected in the trunk kidney, liver, head kidney, skin, spleen, heart and intestine. In response to Aeromonas hydrophila injection, their expressions weresignificantly up-regulated in observed tissues. The expression level of gcC6was high atthe unfertilized egg stage. It was significantly increased at1day post-hatching, but itwas decreased at10days post-hatching. Taken together, our data provided a betterunderstanding of the physiological function of complement C6in vertebrates.2. Linkage analysis of complement C6and C7genes in grass carpFour parental individuals from two families were used to screen the polymorphisms ofcomplement C6and C7genes. A single nucleotide polymorphism loci were found in theforth exon in C6, and another single nucleotide polymorphism loci were obtained in thefifth intron. Finally these two SNPs were used for genetyping the two mapping familiescomprising192individues. The grass carp C6and C7genes were located adjacent toeach other within the chromosome3linkage group between two markers, CID0050andCID0190.3. The polymorphisms of complement C6and C7and their association with resistanceto bacterial hemorrhagic disease in grass carpAfter the challenge with Aeromonas hydrophila, A resource population consisting of186infected individuals and191unfected individuals was constructed. Genomicwalking method was used to obtain the promoter sequences of these two genes, and thesequence length of complement C6and C7were1130bp and1107bp, respectively. Thegenomic sequences of complement C6and C7were9288bp and7068bp, respectively,all consisting of18exons and17introns.Nine SNPs were identified in the genomic sequence of C6. One SNP in exon weresynonymous mutation. Eight of them were sited in introns. We selected the eight SNPsand analyzed the genotype and sllele distribution in susceptible and resistant groups.The statistical results indicated that there was lack of association between C6SNPs anddisease resistance in grass carp. The four SNPs were highly linked each other includingG2638A、G2804C、A3519C、T3591C (r2>80). There were thirteen haplotypes inresource population that generated with the four associated SNPs. haplotype GCCC wassignificantly associated with susceptability to bacterial hemorrhagic disease (p <0.05).Seven SNPs were identified in the genomic sequence of C7. One SNP in exon weresynonymous mutation. One SNP was found in promoter region. Five of them were sited in introns. We selected the six SNPs and analyzed the genotype and sllele distribution insusceptible and resistant groups. The statistical results indicated that C1931T was foundto be associated with disease resistance in grass carp ((p=0.045and p=0.028)). Thethree SNPs were highly linked each other including A-69C、T819C、A2456T (r2>85).There were eight haplotypes in resource population generated with the three SNPs. Nohaplotypes were strongly associated with resistance to hemorrhagic disease (p>0.05).