Cloning, Chromosomal Localization,Prokaryotic Expression And Association Analysis Of The Porcine Sar1b Gene

Sar1 (secretion-associated and Ras-related) functions as a molecular switch to control the assembly of protein coats of COP II -coat vesicles. In human , it has been found that Sarlb is essential for the intracellular transport of chylomicrons from endoplasmic reticulum to Golgi apparatus, and mutations in Sarlb are associated with lipid absorption disorders. In this study, Sarlb gene is selected to be researched as a candidate gene which impacting fat deposition in the pig. It lays an important basis on further study of the Sarlb gene on the impaction of fat deposition through its cloning, chromosomal localization , expression pattern analysis, prokaryotic expression, polymorphism identification and association with production traits, providing a molecular genetic foundation on its application in the molecular marker assisted selection (MAS) in the future. The main results are as follows:1. The full-length sequence of coding region of the porcine Sarlb gene was cloned using the pig specific primers which were designed from the pig EST contig obtained by the method of bioinformatics and comparative genomics according to the corresponding human cDNA sequence. The sequence isolated shows 93% and 91% sequence identity to the corresponding sequence of human and mouse respectively. It has been deposited in GenBank database and the GenBank accession number is AY819557.2. The somatic cell hybrid panel (SCHP) was used for determining the chromosomal regional location of the pig Sarlb gene. The data of PCR results was analyzed using the mapping analysis tools available on the web server. The result of the data analysis indicated that Sarlb gene was mapped to the region of SSC2 (1/2 q24)-q29 (probability of localization equals 0.8696 and error risk less than 0.5 %).3. The INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel was applied for detecting the precise localization of the Sarlb gene. The data of PCR results was analyzed using the mapping analysis software available on the web server. The result of the data analysis revealed that Sarlb gene most closely linked to the gene of IL4 (LOD=8.37) and the nearest microsatellite marker was SW1879 (LOD= 6.68).4. Gene tissue distribution was analyzed by the semi-quantitative RT-PCR in 8different tissues of liver, small intestine, stomach, heart, lung, spleen, muscle, and adipose from a mature Chinese indigenous Exi Black Pigs (Hubei Province, China). The result of RT-PCR displayed that Sarlb gene was expressed in all eight detected tissues. In the tissues of muscle and fat, it was expressed relatively higher.5. Recombinant prokaryotic expression vector pET28a-Sarlb was constructed and it expresses the porcine Sarlb protein successfully by the detection of SDS-PAGE and Western-blot.6. PCR-SSCP was employed to detect a base pair deletion in the 3' untranslated region (UTR) of the Sarlb gene where it was 7 bp (base pair) distant from the last base of stop codon. The polymorphism for the deletion mutation was analyzed by PCR-SSCP among the different pig groups, and allele frequencies and the allele distribution were determined among these populations.7. The polymorphism for the deletion mutation in the 3' UTR of the Sarlb gene was analyzed in the experimental pig population constructed by our lab, including Tongcheng, Landrace & x (Large White x Tongcheng) $ and Large White$ x (Landrace x Tongcheng) ï¿¡. The association of the genotypes of deletion mutation site with some production traits was analyzed using the GLM (Generalized Linear Model) procedure of the SAS (Version 8.2) package. The preliminary results indicated that the mutation is associated with the muscle pH value (P=0.04), and the muscle pH value of pigs with AA genotype was significantly higher than that of pigs with the AB genotypes (P=0.012).