Isolation, Mapping, Spatio-temporal Distribution And Association Of 5 Porcine Genes Related To NADH Respiratory Chain And SMTN Gene

Swine is the most dominating domestic animals feeding for meat production in the world. The meat production performance is the investigative emphasis of investigators including our lab at all times. We found 6 genes differential expression in the skeletal muscle of porcine fetus when we study the porcine genome transcription profile in different breeds and different developmental stages using LongSAGE. 5 among the 6 genes are related to the function of NADH respiratory chain of mitochondrion. As a kind of active organ of metabolism, skeletal muscle contains abundant mitochondrion. Since mitochondrion is the primary organelle productting energy in cells, the genes related to the function of mitochondrion would be associated with the growth and quality of skeletal muscle. However, the study of genes related to mitochondrion is focused on E.coli, cattle and human, and there has no report on swine. Therefore, we selected the 5 genes, as well as SMTN gene related to human cardiovascular diseases, for chromosomal mapping, CDS cloning and analysis, spatio-temporal distribution, mutation riddling, and association analysis with production traits of 3 porcine populations. We expect to know the structure and function of these 6 genes primarily, and supply data for porcine marker assistant selection of improving meat production. Meanwhile, the results of association analysis of 3 pint-sized swine may supply valuable genetics data of germ plasm characteristics for the study of animal model of human disease.The results of this study as follows:1. The 6 genes are isolated using porcine EST information, namely NDUFAB1, NDUFS2,NDUFS3,NDUFS5,NDUFS7 and SMTN gene, the genome DNA segments of 6 genes are cloned and the sequence analysis are completed.2. The 6 genes are mapped on porcine chromosome using IMpRH respectively, NDUFAB1 mapped on SSC3p, linked with marker S0174, the LOD value 10.46; NDUFS2 mapped on SSC4q, linked with marker SW589, the LOD value 8.52; NDUFS3 mapped on SSC2p, linked with marker SW1450, the LOD value 13.46; NDUFS5 mapped on SSC6q, linked with marker SW1473, the LOD value 11.14; NDUFS7 mapped on SSC2q21-q24, linked with marker SW395, the LOD value 7.43; SMTN mapped on SSC14q21-q22, linked with marker SW2612, the LOD value 12.56. The results of mapping are consistent with the results of human corresponding genes.3. The CDS of 6 porcine genes are obtained. The structure and function of coding products of 6 genes are predicted according to the CDS, and the phylogenetic relationship of 6 genes are analysed among swine, human, cow and rat. The results show that the CDS length of NDUFAB1 is 471bp, NDUFS2 1392bp, NDUFS3 795bp, NDUFS5 321bp, NDUFS7 648bp, and SMTN 2475bp respectively.4. By semi-quantitative RT-PCR, the expressive level of 6 porcine genes are studied in skeletal muscle, heart, liver, brain, spleen, lymph node, lung, kidney, large intestine, small intestine, fat tissue of grown pigs, and the skeletal muscle of 33d fetuses, 65d fetuses, 90d fetuses, and grown pigs. The results indicate that the expressive levels of 5 mitochondrion genes are higher in heart, liver, brain and kidney, while lower in skeletal muscle. The expressive level of SMTN gene is higher in spleen, whereas lower in skeletal muscle, heart, brain and kidney. The changing trend of expressive level of 6 genes is similar in different stages skeletal muscle, that is, the expressive level is higher in the skeletal muscle of 65d fetuses, lower in 90d fetuses, very low in 33d fetuses and grown pigs. This improves that 65d post coitus is the most active stage of differentiation of muscle fibre, with higher expressive level of genes, and then the expressive level tends to lower.5. The single nucleotide polymorphism of NDUFAB1,NDUFS3,NDUFS5 and SMTN gene are detected with DHPLC, EST aligning and sequencing. A G/A nucleotide mutation of NDUFAB1was found, which changed the cutting site of MspA1I. A G/A nucleotide mutation of NDUFS3 was detected with DHPLC in an exon region, which leads to the change of amino acid. A G/A nucleotide mutation of NDUFS5 was found in the 5'-UTR, and a C/T mutation of SMTN in an exon.6. The genotype frequency of 4 genes are detected in the pig populations of Tongcheng, White×Landrance×Tong, Large White, Landrance, Guizhou, Wuzhishan and Bama with DHPLC and enzyme cutting. The GG genotype frequency of NDUFAB1 is 0.526, GA 0.428, and AA 0.046 respectively. The frequency of allele A is higher in Large White pig population, whereas very low in Tongcheng and Landrance pig populations. The AA genotype frequency of NDUFS3 is 0.699, AG 0.275, and GG 0.026 respectively. The frequency of A allele is higher among every populations. The CC genotype of NDUFS5 is 0.650, CG 0.312, and GG 0.038 respectively. The C allele show preponderant distribution among every population. The conclusion can be drawed subsequently that the genotype of 3 genes shows serious leaning distribution. The TT genotype of SMTN gene is 0.547, TC 0.267, and CC 0.186. There has no TT genotype in White×Landrance×Tong population, no CC genotype in Wuzhishan population, and no CC and CT genotype in Bama population. There are 1 CT genotype sample in Wuzhishan and 3 CC genotype samples. Therefore, the T allele shows preponderant distribution among local breeds.7. The correlative analysis of 4 genes was carried out with General Linear Model of SPSS software. The genotype of NDUFAB1 gene is highly significantly correlative with percentage of ham (Ham), while significantly correlative with intramuscular fat (IMF). The differences are highly significant between GG genotype and the other two genotypes for the two traits, while no significant difference been found between GA and AA for the percentage of ham, and significant difference between GA and AA for IMF. There have no significant correlation between genotype and the other traits, while there have difference between genotypes for average daily gain from birth to market (ADG), backfat thickness (BF), loin-muscle area (LMA), marbling score (MS) and shear force.The genotype of NDUFS3 gene tends to be significantly correlative with BF. There are highly significant difference between AA and GG genotype, while significant difference between AG and the other two genotypes. The genotype has no significant correlation with other traits, but the results of LSD analysis show that there have differences between genotypes for percentage of leaf fat (LFP), percentage of intramuscular fat (IMF), dressing percent (DP), Ham and LMA.There are highly significant correlation between genotype of NDUFS5 gene and Ham, while no significant difference exists between genotypes. The genotype tends to be significantly correlative with days of age at an ideal market weight (Age).The genotype of SMTN gene is highly significant correlative with Ham, while there have differences between genotypes for ADG, IMF, BF, DP and LMA. The results of T-test show that the difference is very significant between 2 genotypes for triglyceride (TG),cholesterol (CHOL),high density lipoprotein cholesterol (HDL-C),low density lipoprotein cholesterol (LDL-C),alanine amiotransferase (ALT),alkaline phosphatase (ALP),γ-glutamyl transpeptadase (GGT),c-reactive protein (CRP) and mean erythrocytes hemoglobin concentration (CHCM), while there have significant difference between 2 genotypes for apolipoprotein-B (APO-B),white blood cell count (WBC),LVC and lymphocyte percent (LYMPH%). Creatinine (CREA) tends to be significant difference. There have no defference between genotypes for the other traits.