Cloning, Character And Polymorphism Of Porcine USF1 And MBF1 Genes

The traditional animal breeding is gradually combined with the molecular breedingsuch as the marker assistant selection, transgene technology and the functionalgenomics, et al. They are all used as the foreland field and become the main directionfor the swine breeding. The use of marker assistant selection combining with traditionalbreeding methods is promoting the progress of genetic improvement in pigs. As moreand more bioinformatics and database are common, they afford the new method toseparating and localization. In this paper, we used the method of the candidate gene andthe EST information with the Comparative Genomics. We carried out the characterassociation and functional domain prediction. The main results are as follows:1. Based on the method of candidate gene, we found the porcine homologous ESTfrom the ESTs. The special primers were designed through the contigs by the ESTs.USF1, MBF1 and USF1/CD a novel transcript of USF1 gene were isolated from LargeWhite and Meishan cDNA. The sequences of USF1 and USF1/CD were submitted tothe NCBI GenBank and assigned the accession numbers of EF 219407 and EF 208923.2. According to cloned cDNA sequence overlapping primers were designed toamplify the genome sequence of USF1 and MBF1. The overlapping PCR products fromLarge White and Meishan were cloned, sequenced, and assembled into respective contig.The USF1 contig is with a length of 5516bp covering the whole DNA sequence ofUSF1 gene.This sequence was submitted to the NCBI GenBank and assigned theaccession numbers of EF 625884. A T-C mutation which makes the region berecognized by AluI in intron 10 was found in two different pig breeds. Partial DNAsequence of porcine MBF1 gene from exon 2 to exon 5 was cloned and was submittedto the NCBI GenBank with accession numbers of EF 625885. Seven SNPs(two SNPs inexon 4, five SNPs in intron 4) were revealed in MBF1 gene. A T-C substitution atposition 42 in exon 4 caused Fsp4HI-RFLP, A T-C substitution at position 81 in exon 4caused TaqI-RFLP.3. Expression profiles of USF1, USF1/CD and MBF1 genes were analysis bysemi-quantitative RT-PCR assay. They widely expressed in tissues of heart, liver, spleen,lung, kidney, small intestine, stomach, backfat, longissimus dorsi muscle, ovary, uterus.4. The profiles of functional domain, hydrophobicity and hydrophilicity in USF1,USF1/CD and MBF1 protein were estimated and displayed primarily. The multiplesequence alignments were carried out by the CLUSTAL W program. Polygenetictree ofUSF1 and MBF1 protein in different species were analyzed by the CLUSTAL W method in DNAStar software.5. Studied on polymorphic sites of USF1 gene in intron 10 and MBF1 gene inexon 4 by PCR-RFLP, genotype and allele frequencies among different pig populationswere determined. Analysis of PCR-AluI-RFLP in USF1 gene showed that allele Awas predominant in foreign pig breed, with a frequencies of 1.00 for allele A in largeWhite and of 0.85 in Landrace; however allele B was predominant in Chinese pigbreeds, frequencies of allele B in Meishan and Tongcheng was 0.79 and 0.83respectively. Analysis of PCR-TaqI-RFLP in MBF1 gene showed that allele A waspredominant in foreign pig breed, with a frequencies of 0.81 for allele A in large Whiteand of 0.86 in Landrace; however allele B was predominant in Chinese pig breeds,frequencies of allele B in Meishan and Tongcheng was 0.94 and 0.71 respectively.Association with PCR-AluI-RFLP in USF1 gene genotypes and phenotypes ofeconomic traits in 421 F₂(L×M) pigs revealed that USF1 gene affected ratio lean meatversus fat meat, dressing percentage, average backfat thickness, loin eye width, loin eyearea, water content (m.longissimus Dorsi, LD) significantly (p＜0.05, the same below)and affect lean meat percentage, loin eye height highly significantly (p＜0.01, the samebelow). Additive effects of dressing percentage, average backfat thickness, loin eye areawere significantly, Additive effects of loin eye width, loin eye height were highlysignificantly. Dominance effects of lean meat percentage, ratio lean meat versus fatmeat, loin eye area,, loin eye height were significantly.Association with PCR-AluI-RFLP in USF1 gene genotypes and phenotypes ofeconomic traits in 447 F₂(Large Whight×Meishan) pigs revealed that MBF1 geneaffected bone percentage, lean meat percentage, leaf fat weight, backfat thickness atshoulder, backfat thickness at 6-7th thorax, loin eye area, loin eye height, loin eye width,water content (m.longissimus Dorsi, LD) significantly. Additive effects of bonepercentage, backfat thickness at 6-7th thorax, loin eye height, loin eye area weresignificantly. Additive effects of lean meat percentage, backfat thickness at shoulderwere highly significantly. Dominance effects of lean fat weight, loin eye width, watercontent (m.longissimus Dorsi, LD) were significantly.