Isolation, Radiation Hybrid Mapping Of Five Porcine Genes And Their Association With Economic Traits

The establishment of Molecular Biology and its application in pig genetics and breeding make a breakthrough in the animal traditional breeding. In order to provide a basis for the future molecular breeding approach to improve important economic traits of the pig, many groups are struggling to establish high-density gene map (genetic and physical map) and get a full knowledge of the gene structure, function and their association with the economic traits. Recently increasing EST data and the advent of the Bioinformatics bring the investigators new ideas about the study of the genes. Based on the EST data and comparative genomics, the present study in this thesis was to isolate, characterize and physically map the novel genes .The main results are listed as follows:1. The pig specific primers were selected from the pig EST contig constructed according to the corresponding human cDNA. The fragments of five genes were successfully cloned through the PCR amplification from Hubei White pig genomic DNA by using the primes mentioned above. The sequences of three aldolase isozyme genes ( ALDOA, ALDOB and ALDOC) have been deposited in GenBank database with the respective accession numbers: AY359812, AY359813 and AY359811. The other genes isolated were selenoprotein P gene (SEPP1) and membrane protein expressed in epithelial-like lung adenocarcinoma gene (CT120). The comparison of the predicted exonic sequences of the isolated fragment with the corresponding human cDNA was done through the BLAST2 algorithm. The results showed all the fragments harbor the nucleotide sequence similarity more than 80% with the corresponding human cDNA, which showed pig specific fragments of five genes were isolated.2. The INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel was employed to determine the precise location of the five genes. Statistical analysis indicated that the five genes were all significantly linked to the first-generation porcine whole-genome radiation hybrid map (LOD>6.0). The ALDOA gene was closely linked to SW1443 with the LOD score threshold of 6.12. The ALDOB gene was closely linked to SSC1F12 with the LOD score threshold of 17.58.The ALDOC gene was closely linked to SWC23 with the LOD score threshold of 10.8. The SEPP1 gene was closely linked to S0077 with the LOD score threshold of 8.85. The CT120 gene was closely linked to SO106 with LOD score threshold of 11.06.3. Two single nucleotide polymorphisms were found within intron 2 of SEP PI (induced according to the human gene), which just created two enzyme recognition sites for Kpnl and Vspl. The transition (A-G) was found within intron 4 of CTJ20, which also generates an enzyme recognition site for Mspl. The SEPP1 Kpnl-RFLP, Vspl-RFLP and CT120 Mspl -RFLP polymorphism were all demonstrated by PCR-RFLP. The allele frequencies were determined by genotyping the different populations.4. Codominant inheritance of the SEPP1 Kpnl-RFLP and CT120 Mspl-RFLP were demonstrated in the Landrace x (Large Whitex Tongcheng) and Large White x (Landracex Tongcheng) two-generation pedigrees.5. The Mspl-RFLP of CT120 gene was genotyped in the experimental pig populations constructed under the cooperation of our lab and animal husbandry bureau of Tongcheng county, including Landrace, Tongcheng, Landrace x (Large Whitex Tongcheng) and Large White $ x (Landracex Tongcheng) . The preliminary association analysis of CT120 with several immune traits and production traits revealed a significant association between the CT120 and the average backfat thickness (P<0.01).6. The semi-quantitative RT-PCR was performed to determine the expression profile of pig SEPP1 gene in different tissues. The result showed SEPP1 was expressed in live, lung, kidney, spleen and heart. Among the five tissues examined, the mRNA expression level is highest in live, moderate in kidney, spleen and lung, lowest in heart.