Isolation, Identification Of Four Candidate Genes For Skeletal Muscle Growth And Meat Qualtity Traits

Improvement of muscle growth and meat quality has always been one of the main goals for livestock genetics and breeding. As the most abundant tissue in animals, its related growth and development traits are the most important ones to the livestock. In rencent years, with the rapid development of molecular biology and the isolation, identification of several major functional genes related to muscle growth and meat qualities such as RYR1 and RN, the molecular breeding techniques such as marker-assisted selection （MAS） and marker-assisted introgression （MAI） combining with traditional breeding methods have greatly promoted the process of genetic improvement in pigs. The bases for effective MAS rely on the identification of more major genes or markers which are closely connected with the significant economic traits. So in this research, combining the QTLs mapping results, the comparative genome map of pig and human, the differential expression profile between Large White and Meishan pigs of four month old in skeletal muscle and the physiological and biochemical function of candidate genes, four genes were chosen as candidate genes for muscle growth and meat quality traits and studied on. The main results are as follows:1. melusin （Integrin beta 1 binding protein 2）1) Isolation and characterization of full length cDNA sequence. A 1253 bp cDNA were acquired through contig-assembling from EST data and the RACE method, and submitted to GenBank database with DQ002920. Predicted porcine melusin shares high homology （>86%） with that of human, chimpanzee, monkey, cattle, rat and mouse proteins and has the closest genetic relationship with cow.2) Breed specific transcript variant identification. One transcript variant with exon 2 extends from 50 bp to 156 bp and specially expressed in longissimus doris muscle of Large White pigs was isolated. Through RACE method, the 1359 bp in length cDNA sequence which has the maximum ORF extending from 479 bp to 1150 bp and encoding a protein of 223 amino acids was acquired （FJ447492）. The expression pattern in different pig breeds and ages in longissimus doris muscle was also studied by semi quantitative RT-PCR method. 3) Isolation and characterization of full-length genomic DNA sequence. Based on the homology between species and the acquired porcine melusin cDNA sequence, a 5872 full-length genomic DNA sequence of melusin gene was obtained. Comparison analysis with homogenes revealed that porcine melusin gene consists of 11 exons and 10 introns and the location of splice donor/acceptor sites conform to the GT/AG rule.4) Isolation and bioinformatics analysis of promoter. A 797 bp 5’ flanking region of porcine melusin was acquired by conservation comparison between different species. The acquired 797-bp sequence shares 80% and 73% homology with that of human and mouse retrieved by Gene2Promoter. NNPP, MatInspector, CPGPLOT, SIGNALSCAN softwares were used for prediction of promoter, initial position of transcription, distribution of CpG island and transfactos binding to promoter.5) Tissue expression profile anlysis. Tissue expression profile analysis in Large White pigs showed that 2 transcriptional variants were predominantly expressed in skeletal muscle, cardiac muscle, and spleen, weakly in liver and kidney. And only the wild type transcript is detected in lung and ovary, with it highly expressed in lung and weakly expressed in ovary.6) Polymorphism detection. Through sequence comparison of porcine melusin genomic sequences among Meishan pigs, Large White pigs and Landrance pigs, 25 putative SNP sites were identified. Three SNPs denoted G419C, A902G, T3595C can be detected by PstI, RsaI, BstHHI-PCR-RFLP assay respectively. The frequencies of the allele distribution were analyzed in different pure pig breeds. Only haplotype 419C-902G-3595C was detected in Large White and Landrance pigs, whereas the haplotype 419G-902A-3595T was only presented in Meishan pigs, showing this region is in extremely linkage disquilibrium status. SNP detection in the Large White×Meishan F₂ population further confirmed our conclusion.7) Association analysis for different haplotypes. Association analysis of different haplotype with carcass traits showed significant association of the genotypes with BP, RLF, SFT, RFT, TFT, BFT, ABF, LFW, CFW （p<0.01）, LEH, LEW, SP （p<0.05）. Pigs with genotype CGC/CGC trends to have higher LEA and LEW, and lower backfat thickness and LEH. Association analysis of different genotypes with meat quality traits revealed that significant associations of the genotypes with MCV2, MM2, MM1, IMF and WM were observed. Pigs with genotype of CGC have higer MCV2 and WM, whereas pigs with GAT have higher MM2, MM1 and IMF with the mainly genetic effect to be additive.2. APOM （Apolipoprotein M）1) Isolation and characterization of full-length cDNA sequence. A 743 bp cDNA of porcine APOM gene was acquired by EST contig assembling and submitted to GenBank database with DQ329240. Predicted porcine APOM shares high homology （>85%） with that of human, chimpanzee, monkey, cattle, rat, mouse proteins, and has the closest relationship with cow.2) Isolation and characterization of full-length genomic DNA sequence. Based on the BAC colone （BX548169）, a 3621 bp genomic DNA sequence of porcine APOM gene including a 1261-bp 5’ flanking region was obtained. Comparison analysis with homogenes revealed that porcine APOM gene consists of 6 exons and 5 introns and the location of splice donor/acceptor sites in all introns conform to the GT/AG rule.3) Isolation and bioinformatic analysis of promoter. A sequence encompassing the 1261-bp 5’ flanking region and the first exon was subjected to several online sofewares including NNPP, MatInspector, CPGPLOT, SIGNALSCAN to predict the core promoter region, initial position of transcription, distribution of CpG island and transfactors binding sites. One core promoter region with classical TATA box was predicted and the TSS was assigned to 1236 bp.4) Comparative mapping of porcine APOM gene. Based on the extensive synteny between HSA 6p and the p arm and the centromeric region of SSC7, the previously mapped genes surrounding APOM and the recently finished SSC7 reference sequence, the porcine APOM was predicted to lie in SLA-I region in SSC7p1.1.5) Tissue expression profile of porcine APOM mRNA. Semi quantitative RT-PCR results have revealed that porcine APOM is predominantly expressed in liver and kidney, and weakly expressed in fat and ovary with no detected expression in other tissues.6) Polymorphism identification. Through sequence comparasion among Large White pigs, Landrance pigs and Meishan pigs, 16 putative SNP sites were predicted and one mutation site denoted G2289C in intron 2 can be detected by ECO130I-PCR-RFLP assay. Allele G was found to be dominant in Large White and Landrance pigs; while in comparison, the other allele C was found to be dominant in Meishan pigs.7) Association analysis for G2289C mutation. Association analysis of different genotypes with carcass traits showed significant association of the genotypes with BP, SP, SFT, TFT, BFT, ABF, LFW, LEW. Pigs with genotype GG trend to have higher SP, BP, lower backfat thickness and the genetic effect of this loucs is mainly of additive. Association analysis of different genotypes with meat quality traits revealed that only pH （BF） was found to be significantly associated with different genotypes.3.DMPK1) Identification of differentially expressed porcine DMPK gene in skeletal muscle. On Affymetrix Porcine Chip, expression of EST （CO949753） was significantly higher in Large White pigs than in Meishan pigs. Through contig-assembling from EST data, the differentially expressed EST was defined as porcine DMPK gene. Following semi-quantitative and real-time RT-PCR analysis confirmed this result.2) Isolation and characterization of the cDNA sequence. Based on the EST contig information and sequence comparison between different species, a 2673 bp fragment containing a 1874 bp open reading frame was isolated. The encoded protein is 624 amimo acids in length with a calculated molecular mass of 69.2 kDa and an isoelectric point of 4.67. Conserved between species, porcine DMPK is predicted to have a Serine/Threonine proterin kinases catalytic domain and a DMPK coiled coil domain like. It shares 78%, 74% homology with that of human and mouse protein respectively.3) Isolation and characterization of genomic DNA sequence. Based on the homology between species and the acquired porcine DMPK cDNA sequence, a total of 11.2 kb genomic sequence including 1745 bp 5’ flanking region was isolated which spans all the 15 exons. The locations of splice donor and acceptor sites in all introns conform to the GT/AG rule.4) Isolation and bioinformatics analysis of the 5’ regulatory sequence. The isolated 1745 bp 5’ regulatory sequence plus comeplet exonl was subjected to several online bioinformatic analysis software （NNPP, MotifFinder, TFSEARCH, CPGPLOT and SignalScan） to predict potential promoter, initial positon of transhicription, distribution of CpG isand.5) Tissue expression profile analysis of porcine DMPK gene. Semi quantitative RT-PCR experiments showed that DMPK transcripts are expressed predominantly in skeletal muscle, mildly in heart, spleen and fat and weakly in stomach, ovary. 4. G6PT1) Identification of differentially expressed porcine G6PT gene in skeletal muscle. On Affymetrix Porcine Chip, expression of EST （BX672651） was significantly higher in Large White pigs than in Meishan pigs. Through contig-assembling from EST data, the differentially expressed EST was defined as porcine G6PT gene. Following real-time RT-PCR analysis confirmed this result.2) Isolation and characterization of the cDNA sequence. A 1925 bp cDNA of porcine G6PT gene with complete 3’ UTR was acquired by EST retrieval and 3’-RACE method. It contains a 1356 bp open reading frame encoding a protein of 451 amino acids with a calculated molecular mass of 48.7 kDa and an isoelectric point of 8.73. A conserved sugar transporter domain exists in this gene. The putative protein, which shares 95%, 93%, 93% homology with that of human, mouse and rat proteins respectively, has a 22 amino acids insertion mutation between 328 and 329 AA.3) Isolation and characterization of genomic DNA sequence. A total of 5077 bp full-length genomic sequence was isolated. Comparison analysis with homogenes revealed that porcine G6PT gene consists of 9 exons and 8 introns. The locations of splice donor and acceptor sites in all introns conform to the GT/AG rule. 4) Tissue expression profile analysis of porcine G6PT gene. Expression analysis applying semi quantitative RT-PCR assays showed that G6PT transcripts are expressed predominantly in skeletal muscle, mildly in heart, liver and kidney.