Molecular Identification, Polymorphism And Association To Aeromonas Hydrophila Infection Of Major Histocompatibility Complex Class ⅡB For The Giant Spiny Frog, Paa Spinosa

The giant spiny frog(Paa spinosa) is one of the most widely distributed species, mainly distributed in Yunnan, Guizhou, Anhui, Jiangsu, Zhejiang, Jiangxi, Hubei, Hunan, Fujian, Guangdong, Guangxi and Hong Kong. The genus is the basis study of the Ranidae relations and plays an important role in the studying of amphibian evolution and geographic distribution. P. spinosa live in the mountain forest brook with poor adaptability to living environment, especially require high water quality condition, the bacteria exposure in the environment is also an important factor affecting their survival. And due to habitat destruction, over-use of wildlife resources and the spread of global warming caused by bacteria, P. spinosa population have declined by30%. MHC gene is an intracellular factor, the variation pattern is considered to play a key role in the decline of amphibians. The relationship between wildlife MHC gene polymorphism and pathogen infectious has been widely reported. P. spinosa is a highly economic value species, research of its immunity-related MHC genes is very necessary, which can provide a molecular basis to further explore the genetic diversity of P. spinosa.Genes of MHC are obvious candidates for determining disease susceptibility as they have an important role in both the innate and adaptive immune response. P. spinosa reserched in this study has an important economic value to our country, we use the mathod of PCR, RT-PCR, the SMART-RACE to explore of MHC class IIB gene polymorphism and the relationship between polymorphism and resistance to disease, to provide a basis for the screening of MHC IIB disease resistance-related gene molecular markers to establish genetic marker-assisted genetic breeding. The main results are as follows:(1) In the present study, the full length of MHC class IIB cDNA fragment of1,136bp was cloned from the Paa spinosa by homology cloning and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR), ncluding a765bp region encoding for a protein with255amino acid residues, a89bp5’-untranslated region (UTR) and a282bp3’-UTR. Comparison to homologous species which has been published on the NCBI show that the gene sequence obtained is MHC IIB gene sequences and72%similarity with Xenopus. We use MEGA4.0software to analysis the MHC IIB gene sequences of P. spinosa with other known vertebrate MHC IIB genes.(2) For analyzing polymorphism of MHC class IIB cDNA, five wild individuals were used to amplified a fragment of an open reading frame (ORF) encoding244amino acids. A total of eleven unique sequences were detected from84sequences of five samples. Among the five individual, two, one, two, four and one alleles were detected, suggesting that maybe two loci of DAB locus existed in the giant spriny frog. The eleven deduced amino acid sequences were designated as Pasa-DAB*0101-0601, respectively. Among the ORF amino acid sequences,59(24.18%) amino acid positions were variable and43(17.62%) occurred in exon2.(3) An extensive study of polymorphism was examined in80individuals. Genomic DNA from40susceptible and40resistant frog individuals was amplified. Twenty-six different of the paa spinosa MHC class IIB alleles were identified from388clones. Among the63codons,70(37.63%) nucleotide positions and37(59.68%) were found variable. Analysis of all the26sequences shows that the nucleotide substitution happened at45mutation regions. We picked out nine high frequency alleles to study association between MHC class IIB alleles polymorphism and disease resistance/susceptibility. The allele Pasa-DAB*1301and Pasa-DAB*3701(P<0.05) show a statistically significant distribution between dead individuals (Dd) and suvivals individuals (Sv), which might be associate with increased resistance to Aeromonas hydropfila in giant spiny frog. In addition, though no significant, alleles Pasa-DAB*2801was found more in dead individuals than in surviving ones. In this present study, the rate of non-synonymous substitutions significantly exceeds that of synonymous substitutions in MHC genes, the dN/dS ratio is greater than1, which corresponds with the results reported in other species, would be expected if the locus were evolving under positive selection.