MHC Class Ⅱ Gene Polymorphism And Its Association With Disease Resistance, And Full Length Cloning And Expression Of C9Gene In "Whole White" Color Pattern Of Cyprinus Carpio Var. Color

In this research, we first infected the “whole white” color pattern of Cyprinuscarpio var. color artificially with Aeromonas hydrophila, and studied thepolymorphisms of the MHC class IIα and IIβ genes respectively after cloning andsequencing. Then we analyzed the association between gene polymorphism and theability of resistance to Aeromonas hydrophila. On the other hand, the full lengthcDNAof complement component C9gene in “whole white” color pattern of Cyprinuscarpio var. color was cloned by Rapid Amplification of cDNA Ends (RACE), andexplored the expression of C9gene in some tissues that in normal conditions and indifferent time after injection with Aeromonas hydrophila.1. MHC class IIβ allele polymorphism and its association with resistance toAeromonas hydrophilaWe identified84different encoding sequences of the MHC gene within210clones from13resistant and30susceptible individuals for the “whole white” colorpattern of Cyprinus carpio var. color. The sequences were classified into13allelesand Cyca-DAB3*17and Cyca-DAB3*18represented novel genes for this species. Weidentified a total of267and130nucleotide and amino acid variable sites, respectively.They exhibited high variable percentages (42.79%and62.50%). The624bp-longnucleotide fragment included a24bp exon1,276bp exon2,282bp exon3, and42bp exon4, which encoded partial signal peptide, β1domain, β2domain, and partialtransmembrane, respectively. The β1domain possessed a greater number of variablesites than the β2domain. We identified150nucleotide (54.35%) and72amino acid(78.26%) variable sites in the276bp β1domain. In contrast, we only found105(37.23%) nucleotide and54(54.75%) amino acid variable sites in the282bp β2domain. We observed22variable sites within24peptide binding residues (PBR) ofthe β1domain. The values (=dN/dS) were1.366for PBR,0.992for non-PBR inthe β1domain, and0.792for the β2domain, implying that positive selection pressurewas exerted on the β1domain of MHC class IIβ gene of the “whole white” colorpattern of C. carpio var. color. There was a higher percentage of Cyca-DAB3*09inthe resistant individuals (7.81%) than in the susceptible individuals (1.37%, P<0.05).Cyca-DAB3*18was only found in the resistant individuals (3.13%of all individuals).Conversely, Cyca-DAB1*08, Cyca-DAB3*08, and Cyca-DAB3*17were onlyobserved in susceptible individuals.2. MHC class IIα allele polymorphism and its association with resistance toAeromonas hydrophilaThe624bp-long nucleotide fragment of MHC class IIα gene were cloned from43“whole white” color pattern of C. carpio var. color using the specific primer pairDXAF and DXAR. We obtained105different amino acid sequences from the207clones, the sequences were classified into11alleles and Cyca-DXA13、Cyca-DXA14、Cyca-DXA15and Cyca-DXA16represented novel genes for this species. Weidentified a total of214and109nucleotide and amino acid variable sites, respectively.They exhibited high variable percentages (34.29%and52.40%). The nucleotidefragment included a33bp exon1,252bp exon2,285bp exon3, and54bp exon4,which encoded partial signal peptide, α1domain, α2domain, and connecting peptide/partial transmembrane, respectively. The α1domain possessed a greater number ofvariable sites than the α2domain. We identified103nucleotide (40.87%) and52amino acid (61.90%) variable sites in α1domain. In contrast, we only found90(31.58%) nucleotide and49(51.58%) amino acid variable sites in α2domain. We observed17variable sites within21peptide binding residues (PBR) of the α1domain.The values (=dN/dS) were5.186for PBR,0.796for non-PBR in the α1domain,and0.531for the α2domain, implying that positive selection pressure was exerted onthe α1domain of MHC class IIα gene of the “whole white” color pattern of C. carpiovar. color. There were very higher percentage of Cyca-DXA5and Cyca-DXA10inthe resistant individuals than that in the susceptible individuals (P<0.01). Conversely,the percentage of Cyca-DXA8in susceptible individuals was significantly higher thanthat in resistance individuals. Cyca-DXA15was only found in the resistantindividuals (4.62%, P<0.01). Real-Time PCR analysis demonstrated that the liver hadthe highest level in normal conditions, followed by the spleen and muscle, and theintestinal showed the lowest expression level in ten tissues. Moreover, MHC IIαtranscripts were significantly down-regulated in both of liver and spleen afterinjection with A. hydrophila, followed by an increase trend at72-and96-hpost-injection, respectively. In kidney, the expression of MHC IIα was up-regulated at12h-post-injection, followed by a significantly decrease after12h-post-injection.3. Full-length cDNA cloning and expression analysis of complement componentC9geneC9, a component of the Membrane Attack Complex (MAC), plays an importantrole in lyse foreign organisms. In the present study, the full-length cDNA of C9genein “whole white” color pattern of C. carpio var. color was cloned by RapidAmplification of cDNA Ends (RACE). The2158bp-long sequence included a24bp5′-UTR,112bp3′-UTR, and2022bp open reading frame (ORF), which encoded a73.16kDa precursor protein of674amino acids. A signal peptide was deposited in the1-19residues. There was a polyadenylation tailing signal AATAAA at57bp upstreamof3’-UTR PolyA tail. Amino acid sequence analysis showed that C9gene had TSP,LDL-R, MACPF and EGF-like domains that all terminal complement components(TCC) possessed. We also detected28conserved cysteine residues. In addition, wefound a74residues-long glycine-rich region at the256site, which did not exist in anyother reported species except grass carp. Phylogenetic analysis showed that the deduced amino acid sequence of “whole white” color pattern of C. carpio var. colorC9had the highest identity to the C9of Ctenopharyngodon idella with77.5%, and41.8%to44.0%identity to the C9of other teleosts. Semi-quantitative PCRdemonstrated that C9gene was expressed in the liver, spleen and kidney of healthy“whole white” color pattern of C. carpio var. color with the highest level in the liver.Real-Time PCR analysis revealed that C9transcripts were significantly up-regulatedin the liver at12h post A. hydrophila infection, followed by a decrease and an increaseat72h-post-injection, and then decreased at96h-post-injection. In the both of thespleen and kidney, C9transcripts were continually up-regulated, peaked at24h-post-injection, followed by a sharp decrease. Results revealed the importance ofthe C9gene in the immune response.