| Nowadays, it already becomes a tendency for the applications of marker-assistedselection (MAS) by molecular markers to the field of pig breeding. The basis of MASwas to seek the major gene and marker which contributed genetically much to theeconomic traits. With the development of pig genome research, the pig genomicinformation becomes abundant. Especially, QTL mapping of economic trait provides thechromosome fragment which contributed to the target traits. The following researchshould be from QTL coarse locus research to the variation at quantitative trait gene leveland identity the major genes for QTL and study their structure and function which will beused for marker-assisted selection to accelerate the progress of genetic improvement inpigs. Positional candidate cloning is an economic and fast method of gene isolation,which utilizes pig genomic information and seeks gene to study from economic trait QTL.Based on QTL mapping results, gene localization, pig and human comparative map andphysiological or biochemical functions of gene, five candidate genes for economic traitsQTL were selected from pig chromosome 4, 12 and 17. The sequences, structures andfunctions of these genes have not been reported in pig at present. These genes anddifferent transcripts of genes were isolated and identified with comparative genomics andbioinformatics approaches, etc. Association studies of the gene polymorphisms withproduction traits were performed and temporal and spatial expression analyses of thesegenes were investigated, as well as the general study on the promoter of the genes. Themain results are as follows:1. CA3 (Carbonic anhydraseⅢ) gene:(1) A number of pig ESTs were initially identified using the cDNA sequence ofhuman CA3 by running a BLASTN search against the GenBank 'EST-others' databases.These ESTs were retrieved and then assembled into one contig. From this contig, primerpairs were designed and yielded overlapping PCR products. They produced a completecoding sequence of 891bp for pig CA3 of LargeWhite, Landrace and Meishan breeds,which includes 783bp of coding sequence and encodes a 260 amino acid protein andshares 87% identity with human and mouse cDNAs. The cDNA sequence of Meishanbreed has been deposited GenBank under accession number AY789514. (2) According tothe obtained cDNA sequence of porcine CA3 gene, primer pairs were designed to amplifythe DNA templates from three pig breeds. A 9589-bp genomic DNA sequence coveringthe entire coding region of porcine CA3 was amplified. The porcine CA3 gene iscomposed of 7 exons and 6 introns. The nucleotide sequence of Meishan breed has been deposited GenBank under accession number DQ675018. (3) A T/C substitution atposition 363 within intron 1 could be detected as a NcoI PCR-RFLP (restriction fragmentlength polymorphism). Allele frequencies for NcoI PCR-RFLP were studied in unrelatedpigs from eight breeds and allele B was predominant in the Western pig breeds andgenotype AA was fixed in Meishan pigs. There existed two alleles in other Chineseindigenous pig populations. Association analysis in 139 F2 animals showed significantassociations between NcoI-RFLP genotypes and SP, FMP, LMP, RLE LFW, CFW, LEH,LEA, BFT, MCV2 and WM. (4) Comparative sequencing of three pig breeds revealedthat microsatellite SJ160 was identified within intron 5 and SJ158 and a novelmicrosatellite marker which included a tandem repeat of (TC)n were identified withinintron 4. We also detected the allele number and frequencies of the three loci in 7 pigbreeds. For SJ160 microsatellite marker, only two alleles were detected. For SJ158microsatellite marker and the novel microsatellite marker which included a tandemrepeat of (TC)n, only three alleles were detected. (5) The association analysis betweenthe CA3 three microsatellite polymorphisms and product traits in 320 F2 offspring(YorkshirexMeishan) were performed. Statistically significant associations with CL,BFT1 and BFT3 were found at the SJ160 locus. At the SJ158 locus, statisticallysignificant associations with SP, BP, FMP, LMP, DP, LFW, IFR, RNS, BFT4, BFT3 andIMF were found. At the novel microsatellite (TC)n locus, statistically significantassociations with SP, DP, IFR, CL, BFT3, ABF and AST were found, but no significantconclusion can be made on other traits. (6) Expression analysis of CA3 gene wasperformed by Real-time PCR. The porcine CA3 gene was differentially expressed in theskeletal muscle between Yorkshire and Meishan breeds. The expression level of CA3 washigher in the skeletal muscle of Meishan than Yorkshire. During the three stages (1 day,60 day and 120 day) of skeletal muscle development in Yorkshire pigs, we observed thatthe CA3 mRNA expression was up-regulated with age.2. PKLR (Pyruvate kinase, liver and red blood cell) gene:(1) According to the high similar region between human and mouse PKLR cDNAsequences, primer pairs were designed to amplify the DNA templates from three pigbreeds. The partial DNA fragments of PKLR gene of LargeWhite, Landrace and Meishanpig breeds were isolated and the 2376bp nucleotide sequences of the fragments thatinclude the sequences from exon 3 to intron 9 were obtained with cloning and sequencing.(2) A PCR-HaeIII-RFLP developed to detect G/A substitution in exon 4 and PCR-CfoI-RFLP to detect C/T substitution in exon 9. For PCR-HaeⅢ-RFLP, unrelatedindividuals from 5 different pig breeds and the 322 F2 animals in Large White×Meishan resource family were genotyped for population variation investigations and associationanalysis. Results showed that there were three genotypes in all pig breeds with theexception of Exi pigs. This locus was significantly associated with LFW, BFT4, AST andLEH. For PCR-CfoI-RFLP, unrelated individuals from 5 pig breeds and the 124 pigs(Landrace pigs, LargeWhite pigs, LargeWhite×Landrace and Landrace×LargeWhite pigs)were genotyped for population variation investigations and association analysis. Thereexisted three genotypes and genotype BB was fixed in Meishan pigs. Statisticallysignificant associations with BP, BFT3, IMF and WM were found.3. AGL (Glycogen debranching enzyme) gene:(1) The four transcripts of AGL gene of LargeWhite, Meishan and Duroc breedswere isolated by RACE-PCR, genome walking and RT-PCR. The cDNA sequences offour transcripts were 5506bp, 5550bp, 5232bp and 5402bp, respectively. The four formsof mRNA by differential splicing in the 5'-untranslated sequence have same codingsequence of 4599bp and 3'-untranslated sequence of 537bp. (2) A C/T substitution in 5'UTR could be detected as a HpyCH4V PCR-RFLP. (3) A fragment of 1489bp upstreamstart code was obtained by genome walking. Sequencing analysis showed the presence ofcanonical TATA, CAAT and GC boxes. Moreover, a CpG Island of 525bp and manypotential binding sites for transcription factors were identified, including Smad3, Smad4,C/EBPb, MAZF, USF, SP1, AP-2, MEF2, MyoD and HNF-3b/foxa2.4. ENO3 (Muscle-specific enolase) gene:(1) According to the high similar region between human and mouse ENO3 cDNAsequences, primer pairs were designed to amplify the cDNA templates from three pigbreeds by RT-PCR and RACE-PCR. They produced a full-length cDNA sequence of1437bp for pig ENO3 of LargeWhite, Landrace and Meishan breeds which includes1305bp of coding sequence and encodes a 434 amino acid protein and shares 90% and85% identity with human and mouse cDNAs, respectively. The cDNA sequence ofLargeWhite breed has been deposited GenBank under accession number DQ355513. (2)Two different transcripts of ENO3 gene were identified by RACE-PCR and designingprimers on 3' end of exon 1. Differential splicing in the 5'-untranslated sequencegenerates two forms of mRNA that differ from one another in the presence or absence of a142 nucleotide fragment in 3' end of exon 1. The transcript 1 represents the142-nucleotide insert present in the long form of ENO3 mRNA and the transcript 2represents the short form of ENO3 mRNA. RNA secondary structures of 5'UTR of ENO3two transcripts were predicted by the smallest free energy method. There are differentRNA secondary structures for two transcripts. RNA secondary structure of transcript 1 is more stable than transcript 2. It may be result in the low expression level of transcript 1.(3) According to the obtained cDNA sequence of porcine ENO3 gene, primer pairs weredesigned to amplify the DNA templates from LargeWhite, Landrace and Meishan breeds.A 5376-bp whole genomic DNA sequence covering the entire coding region of porcineEN03 was amplified. The porcine ENO3 gene is composed of 12 exons and 11 introns.The nucleotide sequence of LargeWhite breed has been deposited GenBank underaccession number DQ676935. (4) By comparing the sequences of three pig breeds, a Tdeletion mutation at position 405 in intron 9 was detected as a StuI PCR-RFLP showedallele frequency differences among 8 pig breeds. Allele A was predominant in the Chineseindigenous breeds. Allele B was predominant in the Landrace and Duroc pig breeds. Theassociation analysis between StuI-RFLP and product traits in 132 pigs (Landrace pigs,LargeWhite pigs, LargeWhite×Landrace and Landrace×LargeWhite pigs) and 138 F2offspring (Yorkshire×Meishan) were performed. This locus was significantly associatedwith FMP, BFT, MCV, MM1, MM2 and IMF. (5) Using Real-time PCR, we carried outthe temporal and spatial expression analyses of ENO3 two transcripts in different tissues,different pig breeds and different development stages. The transcript 1 was highlyexpressed in liver and lung and only a very low level of expression was detected inmuscle, fat, heart, kidney, stomach, uterus and small intestine. The transcript 1 mRNAlevel was undetectable in spleen. The porcine ENO3 transcript 2 expression levels washigh in skeletal muscle and heart, with medium levels in fat, lung and stomach, with lowlevels in liver, spleen, kidney, small intestine and uterus. An expression pattern was alsoanalyzed in the skeletal muscle of both Western Yorkshire and Chinese Meishan pigs atday-60. The two transcripts were differentially expressed in the skeletal muscle betweenYorkshire and Meishan. The expression level of transcript 2 was higher in the skeletalmuscle of Yorkshire than Meishan; in contrast, the transcript 1 was highly expressed inthe skeletal muscle of Meishan. During the three stages of skeletal muscle development inYorkshire pigs, we observed that the ENO3 transcript 1 mRNA expression was downregulated and the expression level of transcript 2 were shown to be at their highest levelsin skeletal muscle of postnatal 1 day and decreased day-60 and then was up-regulatedwith age. The expression level of the transcript 1 was lower in all of skeletal muscle andother tissue samples than transcript 2, with the exception of liver and kidney. (6) Weproduced a consensus promoter sequence of 1090bp up-stream to the transcriptioninitiation site by genome walking. Sequencing analysis showed the presence of canonicalTATA, GC boxes, CpG Island of 388bp and many binding sites for transcription factors,including MEF3, MEF2, AP-1, Sp-1, GATA-1, EGR2, USF, N-Myc, STAT, MyoD, AREB6, MAZ and MZF1. (7) Progressive deletion constructs in the pig ENO3 promoterregion were created by PCR. For the deletion analysis, ENO3 promoter constructs weretransfected into IBRS-2 pig kidney cells and were analyzed their activities. Constructp(-139, +108) showed no luciferase activity. Deletions from -964bp to -794bp and from-536bp to -355bp decreased ENO3 promoter activity and deletions from -794bp to -536bpand from -355bp to -260bp increased the activity. These results indicate the constructp(-139, +108) has no promoter activity but the 5' flanking region including up to -260relative to +108 contains all the elements necessary to achieve basal promoter activity.Moreover, important positive regulatory elements reside between -794bp and -536bp andbetween -355bp and -260bp, whereas negative regulatory elements lie between -964bpand -794bp and between -536bp and -355bp.5. FOXA2/HNF3B (Hepatic nuclear factor-3-beta) gene:(1) Based on the BLAST result of comparing the nucleotides of human FOXA2 geneagainst pig EST database in NCBI, a set of high matched pig ESTs were assembled intocontig. From this contig, primer pairs were designed and yielded overlapping PCRproducts. They produced a complete coding sequence of 1891bp for pig FOXA2 ofLargeWhite and Meishan breeds which includes 1374bp of coding sequence and encodesa 457 amino acid protein. (2) According to the obtained cDNA sequence of porcineFOXA2 gene, primer pairs were designed to amplify the DNA templates fromLargeWhite and Meishan breeds. A 3925-bp whole genomic DNA sequence covering theentire coding region of porcine ENO3 was amplified. The porcine FOXA2 gene iscomposed of 3 exons and 2 introns and its genomic structure shares the higher identitywith human FOXA2 transcript variant 2. (3) A C/G substitution at position 55 in intron 2could be detected as an AvaI PCR-RFLP. (4) FOXA2 transcript levels were highest inlung, with lower levels in testicle, embryo, liver, spleen, kidney and small intestine,while levels in muscle, fat, heart and stomach were barely detected.
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