Identification, Mapping, SNPs Detection And Association Analysis Of The Porcine Proteasome Activator PA28 And PA700 Related Genes

Pig breeding is always focus on improving the lean percentage, increasing the growth rate et al over the past long periods. However, seeking the high performance traits merely decreased the disease resistance traits because the many disease resistance and performance traits present the negative correlation. The heath ware, disease resistance and the quality have been now paid more attention on the pig breeding and production because the healthy and vigor pigs would decrease the drug use and drug retention, increasing the market competition. So, the researchers want to explore the molecular mechanism of diseases, therefore to improve the disease resistance using the genetic methods.Proteasomal activator PA28 and PA700 gene family is the important research hot point, not only because these genes are play the key role in the antigen presentation mediated by the MHC class I, but also they are related to the immune capacity, oncogenesis and the disease resistance. Combining of the in silico cloning and the biotechnology, the 12 genes, which include 2 genes of the PA28 activator and 10 genes of the PA700 activator, were cloned and physical mapped. The main results are as follows:1. Combining the EST data and the Rapid Amplification of cDNA ends (RACE) method, full-length cDNA of the pig PSME1 and PSME2 gene were isolated and identified. Both sequences has been submitted the GenBank database, the accession number are AF527990 and AF527991. Using the PCR, the full-length genomic DNA sequences of the PSME1 and PSME2 genes were cloned and the sequences were analyzed. These two genes have the similar gene structure, both with the 11 exons and 10 introns.2. The reverse transcriptase - polymerase chain reaction (RT-PCR) revealed that porcine PSME1 and PSME2 genes expressed in all 8 tissues studied. The bands obtained in small intestine and kidneys were weak in both cases, relatively.3. Combining the in silico cloning, RACE and RT-PCR method, the full-length cDNA sequence of PSMC1, PSMC4 and PSMC5 gene and the entire coding region of the PSMC2 and PSMD4 genes were cloned. The amino acids were also been deduced and the motifs were been predicted.4. The genomic DNA fragments of PSMC1, PSMC2, PSMC3, PSMC4, PSMC5, PSMC6 PSMD2, PSMD3, PSMD4 and PSMD5 genes were cloned using the method of PCR. The sequences of these 10 genes were used in the BLAST searches against the 'nr' database and the results showed that all the amplified sequences from pig DNAcorresponded to the gene expected. Of which, the sequences of 7 gene has been deposited to GenBank and got the accession numbers. Full-length genomic DNA sequence of pig PSMC5 gene has been isolated and this gene comprised by 12 exons and 11 introns. All the splice sites of the exon/intron conformed to the GT/AG rule.5.The pig X rodent somatic cell hybrid panel (SCHP) was used for regional assignment of both genes and the INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel was employed to determine the precise location of both genes. Statistical analysis revealed that both genes are located to the pig chromosome 7q 12-23 and closed to T cell receptor factor a (TCRA) with LOD scores of 10.82 and 11.65 respectively.6. 10 genes of PA700 gene family described above were physical mapped by the RH panel. Results showed that these 10 gene distribution on the 8 different pig chromosome. PSMC1 gene was located on SSC7, PSMC2 gene was mapped to SSC9, PSMC3 and PSMC4 genes were mapped to the SSC2pl6 and SSC6qll-ql2, respectively; Both PSMC5 and PSMD3 gene were located to the SSC12p; PSMC6 and PSMD5 gene were located to the SSClq; PSMD2 gene and the PSMD4 gene are assigned to the SSC13q41 and SSC4q21-23 respectively. All the results were creditable due to the LOD scores.7. Identification for mutations in the amplified fragments of these 12 genes (including 2 genes of PA28 and 10 genes of PA700 genes) were performed and the 10 SNPs were detected by PCR-RFLP, these loci are as follows: PSME1 gene Sphl-RFLP (560C-560T, intron8), PSMC2 HpaII-RFLP (279G-279C, intron...