Isolation And Functional Analysis Of Differentially Expressed Genes In Liver Tissue Between Large White And Meishan Pigs

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.Due to the difference of origin,genetic background,local environment and artificial selection between Chinese indigenous and foreign commercial pig breeds,they have different intrinsic features.Meishan pigs（Chinese indigenous pigs） have lower lean meat content in their carcasses compared to Large White pigs（Western pigs）,but the lean meat of Meishan pigs is of better quality.Liver is the most important organ in the procedure of substance metabolism of organism.Glycometabolism,lipid and protein metabolism were occurred in the liver.Phenotypic variance between the Large White and Meishan pigs may be related with substance metabolism which occurred in the liver.Thus,mRNA differential display technique was used to isolate and identify the differentially expressed genes in livers between Large White and Meishan pigs in the present study,and the gene functions were also primarily analyzed.Porcine differentially expressed genes SMNDC1（Survival motor neuron domain containing 1）,HOXA5 （Homeobox A5）,TIAF1（TGFB1-induced anti-apoptotic factor 1）,MYO18A（Myosinâ…©â…§a）,POLDIP2（Polymerase（DNA-directed）,delta interacting protein 2） and PHKG2 （Phosphorylase kinase,gamma 2） were choosen for further analyses of the potential functions.These main results were as follows:1.The gene expression was analyzed in liver between 60 days old Large White and Meishan pigs by mRNA differential display.We also observed nearly 3000 bands in differentially displayed PAGE gel,and almost 1000 ones can be repeated in duplicate PCR.2.There are 70 differentially displayed ESTs in PAGE gel.Most ESTs were not homologous to the sequences in GenBank database.The result of validation showed that a majority of ESTs were really differentially expressed,while a few of them weren’t by semi-quantitative RT-PCR.3.The differential expression of the six genes between Large White and Meishan pigs were further identification by real-time quantitative PCR and semi-quantitative RT-PCR.The result showed that the mRNA expression level of HOXA5,TIAF1, MYO18A and POLDIP2 gene was higher in Meishan pigs than Large White pigs, whereas the mRNA expression level of SMNDC1 and PHKG2 gene was more abundant in Large White pigs than Meishan pigs.4.We cloned the cDNA sequences of six genes by electronic cloning,comparative genomic technology and rapid amplification of cDNA ends（RACE）.（1） SMNDC1, obtained cDNA sequence 1875bp,the ORF is 717bp and encodes 238 amino acids, GenBank accession number EU571478.（2） HOXA5,obtained cDNA sequence 1395bp,the ORF is 813bp and encodes 270 amino acids,GenBank accession number EU₀24116.（3） TIAF1,obtained cDNA sequence 1633bp,the ORF is 348bp and encodes 115 amino acids,GenBank accession number EU872207.（4） MYO18A, which has two isoforms,MYO18A₁（6231bp） and MYO18A₂（6186bp）,the difference of the two transcripts is the exon 39.（5） POLDIP2,obtained cDNA sequence 2410bp, the ORF is 1107bp and encodes 368 amino acids.（6） PHKG2,obtained cDNA sequence 1469bp,the ORF is 1221bp and encodes 406 amino acids,GenBank accession number EU169240.The DNA sequences of these genes were obtained and the genomic structures were analyzed.All splice sites of exon/intron of these genes conformed to the GT/AG rule.5.Using DNAStar,CLUSTAL W and some other related software,we analyzed the gene structure,protein structure and conserved motifs of these six genes.In addition, the corresponding phylogenetic trees were constructed.6.In order to study potential functions of target genes,temporal and spatial expression profiles were analyzed using real-time PCR.It was demonstrated that during seven stages of skeletal muscle development in Large White pigs after birth,the mRNA expression levels of the SMNDC1 gene decreased gradually with age.But in Meishan pigs,the SMNDC1 gene mRNA expression was up-regulated from postnatal 3 days to 150 days.The mRNA expression levels of the HOXA5 gene decreased gradually with age and the TIAF1 gene had no significant differences during the skeletal muscle development.The tissue distribution of the six genes in heart,liver,spleen, lung,kidney,stomach,small intestine,uterus,ovary,backfat and longissmus drosi indicated that they were expressed in most tissues and displayed different expression patterns.7.Single nucleotide polymorphisms（SNPs） in the six genes were detected.It was demonstrated that the HOXA5 gene contained one SNP for PCR-RFLP （C1817A-Bpu1102 I-RFLP）,which was located in the intron among three potential polymorphism sites following screening the genome DNA sequence.The TIAF1 gene contained a 6 base-pair deletion mutation for PCR-RFLP（PCR-Eco47 I-RFLP）, which was located in the exon among five potential polymorphism sites.The POLDIP2 gene contained one SNP for PCR-RFLP（C306A-BsuR I-RFLP）,which was located in the 3^th exon among six potential polymorphism sites.The PHKG2 gene included three SNPs for PCR-RFLP（G785A-Mst I-RFLP,G866A-Tag I-RFLP and G875A-Pst I-RFLP）,which were respectively located in the 8^th（for the first two sites） and 9^th exons.8.Genotyping of a total of six polymorphic locus showed that there are abundant polymorphisms in various pig breeds.Association analysis was performed between polymorphisms and important product traits in Large White×Meishan F₂ offspring, and the results showed:（1） For C1817A-Bpu1102 I-RFLP polymorphism of HOXA5 gene,significant effects were observed on LEA（P＜0.05）;（2） There are significant difference between TIAF1-Eco47 I-RFLP polymorphism and CL（P＜0.05）,MM1 and IMF（P＜0.01）;（3） For C306A-BsuR I-RFLP polymorphism of POLDIP2 gene, significant effects were observed on CL（P＜0.01）,SFT and AFT（P＜0.05）;（4） For the PHKG2 gene,highly significant associations in the G785A（PCR-Mst I-RFLP） site was detected between genotypes with LMP（P＜0.01） and significant association between genotypes with IFR（P＜0.05）.In the G866A（PCR-Taq I-RFLP） site,there were highly significant association between genotype with RLF（P＜0.01） and significant association with LMP,IFR,SFT and AFT（P＜0.05）.In the G875A （PCR-Pst I-RFLP） site,there were significant association with LMP（P＜0.05）.9.Genome PCR walking technique was used to clone the proximal promoter region of porcine SMNDC1 gene,and we obtained a fragment of 1242bp.Bioinformatics approaches were adopted to analyze the proximal promoter region of porcine SMNDC1 gene,and the putative binding sites of transcription factors in the regulatory region of the gene were analyzed by online software.10.To determine the location of the promoter activity of porcine SMNDC1 gene,we constructed 10 recombinants of progressively 5’-deleted DNA fragment linked to the pGL3 reporter,respectively.These recombinants were transiently transfected into PK15 cells.Transcriptional activity of SMNDC1 recombinants normalized by Renilla was significant difference with pGL3-Basic expect for construct 931 and 349 （P＜0.05）.Construct 688 contains highest activity and 3 times reduction of transcriptional activity to 349 indicated that negative regulation factors located probably in this region.From the 349 to 109 enhanced the promoter activity by 5.5-fold indicated that positive regulation factors located probably in this region.