| With the development of porcine genomic studies, many genes associated with pig production traits have been identified, however, additional genes need be identified and further characterized. Moreover, the number of porcine muscle fibers, fixed at birth, is an important factor affecting animal meat quality and quantity, which positive associations exist between muscle fiber number and average daily gain and growth rate. Therefore, it will be necessary for isolation and identification of these genes associated with econimic traits from the fetal cDNA library of porcine skeletal muscle.In this present study, porcine 15 genes were isolated from skeletal muscle cDNA library of pig day-55 embryos, and CA3 and HUMMLC2B genes were choosen for further analyses of the potential functions. These main results were as follows:1. Combining sequencing of the cDNA library and porcine EST data, porcine 15 genes were isolated and identified, and these genes were: K-ALPHA-J, TUBB, MAPRE1, TUBA3, ATP6VOE, RPL6, RPL7, RPL10, HUMMLC2B, BNIP3L, NHP2L1, CA3, CCNT2, PC4 and FLJ30294. Sequences of cDNA and predicted proteins of these genes (except for CCNT2 and FLJ30294) were further analyzed.2. These above genes (except for HUMMLC2B) were assigned in pig×hamster hybrids panel, and the results were followed: K-ALPHA-1: SSC5p, TUBB: SSC7p11, MAPRE1: SSC17q21-23, TUBA3: SSC5p11-pl5, RPL6: SSC14q22-24, RPL7: SSC4q11-14, RPL10: SSCXq26, ATP6VOE: SSC16q, BNIP3L: SSC14q21-22, CCNT2: SSC15q12-14, NHP2L1: SSC5pl2-15, FLJ30294: SSC3q11-14, PC4: SSC16q11-14 and CA3: SSC4q11-q14. CA3 was further mapped using porcine somatic cell hybrid panel.3. To the CA3 gene, two different cDNA sequences (namely CA3b and CA3a) were obtained by sequencing of the cDNA library and e-PCR of porcine ESTs sequences, respectively. A gap was shown between the cDNA sequences of CA3b and CA3a by sequence comparison, and the predicted proteins showed that CA3b was the absence of 78 amino acids in the C-terminal domain of CA3a. The fluorescence mapping result using confocal microcopy showed the GFP-fused CA3a protein was distributed throughout the whole cell, but the GFP-fused CA3b protein was only distributed in cytoplasm.4. The full-length DNA sequences of the CA3 and HUMMLC2B genes were obtained and the genomic structures were analyzed. It was identified that the CA3 gene comprised 7 exons and 6 introns (for the cDNA sequence of CA3a), which spaned about 10.5 kb. The HUMMLC2B gene comprised 7 exons and 6 introns, which spaned about 2.9kb. All splice sites of exon/intron of the two genes conformed to the GT/AG rule.5. In order to study potential functions of target genes, temporal and spatial expression profiles were analyzed using real-time PCR. It was demonstrated that the expression levels of CA3 increased from prenatal 33-day-old to 65-day-old skeletal muscle in Tongcheng pigs. These levels subsequently decreased to similar levels as those seen in prenatal 90-day-old pigs, as well as those in postnatal 2-day-old, 28-day-old, and adult pigs, but the expression patterns of Chinese Tongcheng pig embryos were different from that of Landrace pig embryos. CA3 was expressed at higher levels in skeletal muscle and liver than lung, lymph node, large and small intestine, but was not detected in heart and spleen in an adult Wuzhishan pig.6. The results revealed HUMMLC2B expression variation in a waveform manner in the skeletal muscle of both Chinese Tongcheng and Western Landrace pig breeds at days 33, 65 and 90 post coitum (p.c). After birth, the expression levels of HUMMLC2B were also found to decrease gradually with age. Our spatial expression analysis showed that HUMMLC2B was highly expressed in the semitendinosus, gastroenemius, biceps femoris and longissimus dorsi muscles. In contrast, only low levels of expression of this gene were evident in fat, and no expression was detectable in brain, heart, kidney, lung, liver, lymph node, spleen, stomach, or in either the large or small intestine.7. Construction of GFP-fused HUMMLC2B expression vector was transfected into PK15 cells, and the fluorescence mapping result using confocal microcopy showed that the protein was distributed throughout the whole cell containing cytoplasm and nucleus.8. Single nucleotide polymorphisms (SNPs) in the CA3 and HUMMLC2B genes were detected. It was demonstrated that the CA3 gene included two SNPs for PCR-RFLP (BsuRI-PCR and HinfI-PCR), which were respectively located in the 5th and 6th introns. The HUMMLC2B gene contained four PCR-RFLP sites (two Hin1I-PCR, MspI-PCR and BglI-PCR), which were respectively located in the 3 th intron, 4th exons and 5th introns (for the last two sites) among twenty-three potential polymorphism sites following screening the HUMMLC2B genome DNA sequence. Additional four SNPs, which were respectively located in the 6th intron (two SNPs) and the 7th exon (two SNPs), were detected using denaturing high performance liquid chromatography (DHPLC).9. Genotypes and allele frequencies of these above six PCR-RFLP sites of the CA3 and HUMMLC2B genes were determined in five Chinese pig breeds, Wuzhishan pigs, Xiang pigs, Bama xiang pigs, Tongcheng pigs and Laiwu pigs, and three introduced pig breeds, Landrace, Yorkshire and Duroc. 10. Associations were analyzed between genotypes of these above polymorphisms of the CA3 and HUMMLC2B genes and phenotypes of economic traits in the experimental population constructed by the cooperation of our lab and animal husbandry bureau of Tongcheng county, which consisted of Tongcheng, Large White, Landrace and and a further pair of three-cross breeds, Landrace×(Large White×Tongcheng) and Large White×(Landrace×Tongcheng). The results were as follows:①For the CA3 gene, highly significant associations in the G8607A site was detected between genotypes with intramuscular fat content (P＜0.01) and significant association between genotypes with percentage of ham (P＜0.05); In the G8371A site, there were highly significant association between the GG genotype of pigs with intramuscular fat content and marbling score and that of the GA genotype (P＜0.01), and significant association with average backfat thickness (P＜0.05).②For the HUMMLC2B gene, no significant association in the G1094A and T1513C sites was detected between genotypes and traits; In the G1876A site, there was significant association between the GG genotype of pigs with dressing percent and meat color and that of the GA genotype (P＜0.05); In the T2005G site, there was significant association between the GG genotype of pigs with meat color and that of the TG genotype (P＜0.05), and between the TT genotype of pigs with total erythrocytes (RBC), hematocrit (HCT) and red cell distribution width and that of the TG genotype (P＜0.05).
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