| MHC gene is a culster of closely chain genes, It widely exists in the vertebrate body and participate in the body’s immune response. previous studies have been reported that the exon 2 of MHC class ⅡB gene with highly polymorphism, and these highly polymorphic alleles may be correlate with disease resistance. The giant spiny frog (Quasipaa spinosa) is one of the most widely distributed species, At present mainly distributed in the region south of the Yangtze river. Human activities destroyed the wild living habitat, but also makes over-use of wildlife resources of wild Quasipaa spinosa, combined with the global warming is caused by the spread of disease, causing the populations of giant spiny frog are declining. To protect the resource of Quasipaa spinosa, the research of MHC genes which related to their immune system is of great significance.To explore the correlation of MHC gene and disease resistance, one hundred individuals by intraperitoneal injection with single cell bacteria (Aeromonas hydropfila) in the way of artificial infection, used specific primers, genomic DNA from fifty-three susceptible and fourty-seven resistant frog individuals was amplified, and acquired the exon 2 sequence for length of 186 bp. We Picked out 402 positive cloning sequencing, obtained 31 different nucleotide sequences with the way of sequence alignment. In the thirty-one sequences, we picked out six high frequency alleles to study association between MHC class ⅡB alleles polymorphism and disease resistance/susceptibility. The 6 different alleles while exists in individual death and survival disease at the same time, but with different frequency. We used spss to analysis the 6 different alleles frequency differences in the two groups, the allele qasp-DAB*h and qasp-DAB*1 show a statistically significant in 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, alleles qasp-DAB*e was found more in dead individuals than in surviving ones. This illustrated it maybe have a relevant with the susceptibility of Aeromonas hydropfila.We put the suvival individuals Natural breeding progeny in farming field. To study the disease resistance and genetic stability of offsprings, we selected one hundred and fifty individuals randomly. The survival rate of offspring individuals were 90%,84% and 80% after they were infected with Aeromonas hydropfila, Acinetobacter Citrobacter and Werkman and Gillen respectively, the survival rate is significantly higher than normal group, and offspring for each of the bacteria resistance does not exist significant differences. By sequencing method to obtain the offspring individual’s MHC ⅡB exon 2, the polymorphism of sequence can be found in almost all parent individuals. The allele qasp-DAB*h and qasp-DAB*l in offspring of 0.56 and 0.47 respectively, significantly higher than the normal individuals. This illustrate the two resistance genes can be stable inherited. Therefore, qasp-DAB*h and qasp-DAB*1 can be used as molecular markers for selective breeding of resistant strains of Q. spinosa.At the same time, we determined some immune related indicators such as the number of leucocytes, red blood cells and the phagocytosis of phagocytes. We found in offspring indviduals the white blood cell count and the phagocytosis of phagocytes is 85%and 11.5×103 respectivley, significantly stronger than the normal individuals. That illustrated the offsprings have stronger immune system. Nonspecific immune is obtained by genetic, offspring individuals may inherit the good qualities of the parents. |
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