Cloning, Expression Of Complement C9 And Perforin Gene In Oreochromis Niloticus And Screening Of Their SNP Resistance To Streptococcus Agalactiae

Tilapia is the dominant species of our country freshwater fish farming. But in the breeding process,frequent outbreaks of Streptococcus agalactiae disease restrict the sustainable development of China’s tilapia industry severely. Currently, There no effective prevention methods to cure the disease, so from the germplasm, breeding anti-streptococcal disease directly, foster anti-streptococcal disease new tilapia varieties(lines), is worth looking forward measures anticipated measures. In this paper, we cloned the Nile tilapia immune-related gene complement C9(OnC9) and perforin(OnPFP), and the expression and structure were analyzed to understand their biological function preliminary. As well as the single nucleotide acid polymorphism were analyzed. And we analyzed different genotypes or haplotypes associated with resistance to Streptococcus agalactiae disease in Nile tilapia. The results obtained as follows:1. Molecular cloning and expression of gene complement C9 and perforin gene in tilapia The primers were designed according to the prediction sequence of C9 and perforin gene, and obtain the part of full-length fragments. RACE method was used to clone full-length cDNA sequences of OnC9 and OnPFP gene. In the present study, a full-length OnC9 cDNA was 2502 bp, containing 1761 bp open reading frame,encoding 586 amino acids. OnC9 amino acid analysis found the presence of conserved domains: TSP1, LDLRA, MACPF, EGF-Like. The deduced amino acid sequence of OnC9 shared 73.0% similarity with Po C9(Paralichthys olivaceus C9),and 49.3%-71.4% identity with the C9 of other teleosts. Two complete cDNA of OnPFP gene were cloned meanwhile, a full-length OnPFP1 cDNA was 2461 bp,containing 1764 bp open reading frame, encoding 587 amino acids. OnPFP1 amino acid analysis found the presence of conserved domains: MACPF and C2. a full-length of OnPFP2 cDNA sequence was 2442 bp, ORF was 1266 bp, encoding 421 amino acids. OnPFP2 has the C2 domain only. The analysis of NJ phylogenetic tree show that the sequence homologies of OnPFP1 and OnPFP2 amino acid was 78.5%, amino acid sequence homology with the other fish of OnPFP1 was 38.2-75.0%, as well as the amino acid sequence homology with the other fish of OnPFP2 was 36.5-78.2%.Real-time quantitative PCR(q PCR) analysis demonstrated that the expression level of OnC9 in different tissues was significantly different, and the highest level was detected in the liver; the lower level was detected in intestine, kidney, gills, skin and muscle; the lowest level was detected in brain, spleen, heart, head kidney, thymus and blood. The OnC9 expression in liver, kidney and other tissues appeared three peak fluctuations at 12 hours, 48(or 36) hours and 120 hours post infection of Streptococcus agalactiae. OnPFP1 and OnPFP2 in the brain, kidney, spleen, heart,liver, gill, muscle, head kidney, skin, thymus, intestines, blood was all expressed, and expressed various amounts in different tissues, OnPFP1 expressed higher levels in the spleen and gill, OnPFP2 expressed in spleen and head kidney in highest level. Within the infection of Streptococcus agalactiae in 216 hours, OnPFP1 expression levels appeared the first rise, then fall, then rise again and decline end in the liver, spleen,gills, kidney, head kidney, thymus. The expression of OnPFP2 increased gradually in the liver, spleen, gills, kidney, and the expression in the thymus is the first to rise, and then decreased.2 Screening of SNPs in complement C9 and perforin gene of Oreochromis Niloticus and their association with resistance to Streptococcus agalactiae disease Cloning genomic sequence of OnC9 和 OnPFP and screening single nucleotide polymorphism locus, and after the challenge Streptococcus agalactiae with artificial methods, we constructed a resource population consisting of susceptible and resistant groups. Acquiring more SNPs through Snapshot method and to study the correlation between these SNP and Streptococcus agalactiae disease resistance.PCR was used to obtain the geonomicDNA of OnC9 and OnPFP genes. The genomic sequences of OnC9 were 6883 bp, consisting of 11 exons and 10 introns. The genomic sequences of OnPFP1 were 6849 bp, consisting of 6 exons and 5 introns and the genomic sequences of OnPFP2 were 3812 bp, consisting of 3 exons and 2 introns.Twenty-two SNPs were identified in the genomic sequence of OnC9. nineteen SNPs in intron. Three SNPs in exon and two of them were non-synonymous mutations, one of them were synonymous mutation. We selected the twenty-two SNPs and analyzed the genotype distribution in susceptible and resistant groups. The statistical results indicated that the genotype frequencies of C-2660 T and T-5790 A sites were significant differences between resistant and susceptible populations(P<0.05). Linkage disequilibrium analysis shows that multiple sites in high linkage(r2>0.80). There were twelve haplotypes in resource population and haplotype GTTAG and ATGA was significantly associated with susceptability to Streptococcus agalactiae disease(P< 0.05).Sixteen SNPs were identified in the genomic sequence of OnPFP1, Three SNPs in exon were non-synonymous mutations. Thirteen of them were sited in introns. We selected the sixteen SNPs and analyzed the genotype and allele distribution in susceptible and resistant groups. The statistical results indicated that the genotype frequencies of C-2660 T and T-5790 A sites were significant differences between resistant and susceptible populations(P<0.05). The three SNPs were highly linked each other including G-2019C、A-3827C、A2154G(r2 = 0.813, r2 = 0.955). There were three haplotypes in resource population generated with the three SNPs. No haplotypes were strongly associated with resistance to Streptococcus agalactiae disease(P> 0.05).Five SNPs were identified in the genomic sequence of OnPFP2, Three SNPs in exon were non-synonymous mutations. two of them were sited in introns. We selected the five SNPs and analyzed the genotype and allele distribution in susceptible and resistant groups. The statistical results indicated that there was lack of association between OnPFP2 SNPs and disease resistance. The two SNPs were highly linked each other including C-690T、C-618T(r2 = 0.83). There were three haplotypes in resource population generated with the two SNPs. No haplotypes were strongly associated with resistance to Streptococcus agalactiae disease(P> 0.05).