MHCⅡ Gene Polymorphism And Its Association With Disease Resistance In Cyprinus Carpio Haematopteus Challenged With Aeromonas Veronii

Cyprinus carpio haematopteus is one of the major freshwater aquaculture fishes in our country, which economic and nutrient values of Cyprinus carpio haematopteus are higher, that plays a very important role in aquaculture production. In recent years, Aeromonas veronii has become one of the main pathogens, which threaten to the healthy development of aquaculture, but there is no effective measure to against outbreak of this bacteria. The basic approach to resolve the problem is to culture fish species with disease resistance to Aeromonas veronii by using molecular marker-assisted selective breeding. Major histocompatibility complex(MHC) is a group of highly polymorphic genes associated with immune found in vertebrates. Due to its high polymorphism among fish, MHC has been attracted much attention to its structure, function, polymorphism, expression and disease resistance. The relationship between polymorphism of the MHC II genes(DAA and DAB) and the disease resistance of Cyprinus carpio haematopteus was analyzed after infected Aeromonas veronii artificially in the present study. Real-time quantitative PCR was used to detect the expression levels of MHC II genes in some tissues that in normal condition and in different time after injection with Aeromonas veronii. This study had identified potential candidate MHC II genes to provide a theoretical foundation and an important basis for improving disease resistance in fish breeding selection. The main results are shown as follows:1. MHCⅡDAA gene polymorphism and its association with disease resistance in Cyprinus carpio haematopteus challenged with Aeromonas veroniiA total of 54 alleles of MHC II DAA gene were identified in 165 clones from 17 resistant and 16 susceptible Cyprinus carpio haematopteus individuals. The 427 bp-long nucleotide fragment encoded for partial signal peptides, complete a1, a2 and partial a3 domains. The mutation probabilities of nucleotide and amino acid were 40.95%(172/420) and 65%(91/140), respectively. Each allele frequency and its subtypes were not distributed equally, Cyca-DAA*0101 and Cyca-DAA*1502 with the highest frequency were 0.1212 and 0.0909. There were four alleles existed in both resistant and susceptible individuals, and the percentage of Cyca-DAA*0101 in resistant individuals was 9.09%, while it was only 3.03% in susceptible individuals,showed significant differences(P<0.01). 29 alleles were only found in the resistant individuals, especially Cyca-DAA*0901(5.45%) with the largest proportion(P<0.01). Conversely, 19 alleles were observed in susceptible individuals, and Cyca-DAA*1502 with the largest percentage of 9.09%(P<0.01). After structure analysis, the ratio of non-synonymous substitutions and synonymous substitutions(d N/d S) were 2.556 and 0.769 in the peptide binding region(PBR) and in the non-peptide binding region(Non-PBR), respectively. It showed that positive selection was predominant mechanism in natural evolution of MHC II DAA gene in Cyprinus carpio haematopteus.The RT-PCR analysis showed that the MHC II DAA gene was expressed in all 9 tissues, with the highest levels in kidney, liver, intestine, and spleen; the moderate levels in heart, gills, and brain; and the lowest levels in muscle and eyes. After infection with Aeromonas veronii, the expression levels of MHC II DAA gene varied in liver, spleen, kidney, and intestine at different time, it indicated that the gene had important effects on the immune response.2. MHCⅡDAB gene polymorphism and its association with disease resistance in Cyprinus carpio haematopteus challenged with Aeromonas veroniiA total of 72 alleles of MHC II DAB gene were identified in 165 clones from 17 resistant and 16 susceptible Cyprinus carpio haematopteus individuals. The 327 bp long nucleotide fragment included partial signal peptides, complete β1 and partial β1domains. There were 221 nucleotides(67.58%) and 104 amino acid(100%) variable sites. Each individual was multi-locus and each locus contained a large number of alleles, the genes with higher frequency were Cyca-DAB*0801(0.151), Cyca-DAB*1001(0.0909), and Cyca-DAB*0701(0.0788). There were 5 alleles found in both resistant and susceptible populations, the percentages of Cyca-DAB*0202 and Cyca-DAB*1001 were significant higher in susceptible population than resistance population(P<0.05), but in resistant population, the frequency of Cyca-DAB*0101 was larger than it in susceptible population, the difference was significant(P<0.01). There were 38 alleles only found in the resistant population, and the differences of each allele frequency were not significant. Conversely, 29 alleles were observed in susceptible population, in which Cyca-DAB*1001(9.09%) with the largest proportion(P<0.01). The ratio of non-synonymous substitutions and synonymous substitutions(d N/d S) in the PBR(2.556) and in the Non-PBR(0.898) suggested that balancing selection was the occurrence mechanism for MHC II DAB genes existence.Real-time PCR results indicated MHCⅡDAB gene expressed in all detected tissues with different expression levels. High expression were detected in kidney, liver, spleen, and gills; moderate expression in heart and intestine; and low expression in brain, eyes and muscle. After infection with Aeromonas veronii, the expression levels of MHC II DAB gene varied in liver, spleen, kidney, and gills, it indicated that the gene had important effects on the immune response.