Isolation And Preliminary Study Of Porcine Fat Deposition Genes PTP-1B And AQP7

In this study, two candidate genes were selected based on QTL mapping results, pig and human comparative map and physiological or biochemical functions, that is PTP-1B and AQP7 genes. The two genes were isolated and sequenced with comparative genomics and bioinformatie approaches, and then make out genetic effect analyses between the polymorphisms and pig important economy traits especially fat deposition traits. And expression patterns of the two genes in pig different tissues were also studied with Real-time PCR. The main results were as follows:1 PTP-1B gene isolation, sequencing and association analysisA number of pig EST sequences were fished using the eDNA sequence of human PTP-1B gene by searching EST database of BLAST. Then Primers were designed based on the one to ampliy parts of swine PTP-1B eDNA including full coding sequences. Then ampliy intron6, 7, 8, 9. The splitting sites conform to GT/AG rules in these introns. There were 14 SNPs in introns and 3 SNPs in 3' untranslated sequences, A A-G mutation locus could be digested by Eco88I. the polymorphim was detected by PCR-Eco88I-RFLP. As a result, it was showed that:Among 22 Meishan, 16 Large White and 21 Landrace pigs, The allele distribution revealed that western pigs Large white, landrace had singularity G allele, While Chinese indigenous breed Meishan mainly on AG genotype. So allele frequencies of this polymorphism are significantly different between western commercial pig breeds Large white, landrace and Chinese native breeds Meishan.Association analysis between the polymorphism and fat deposition traits was performed in 213 Large white×Meishan F₂ pigs. Significant associations of the genotype with fat deposition traits such as leaf fat weight (LFW), internal fat rate (IFR), and Thorax- waist fat thickness were found. The individuals with AG genotype have lowest leaf fat weight and internal fat rate than the others and GG genotype have lowest Thorax- waist fat thickness.2 Porcine AQP7 gene isolation, sequencing and association analysisA pig cDNA library sequences was fished using the cDNA sequence of human AQP7 gene by searching EST database of BLAST. Primers were designed based on the one to ampliy parts of swine AQP7 cDNA. Then partial genomic sequence were obtained, that is intron 1, 4, 5, 6. There were 6 SNPs in introns, A A-G mutation site could be digested by PstI. The polymorphim was detected by PCR-PstI-RFLP. The detailed results are as following:Among 24 Meishan, 21 Large White and 18 Landrace pigs. The allele distribution revealed that western pigs Large white, landrace had higher frequencies of the G allele, While Chinese indigenous breed Meishan had higher frequencies of A allele than G. So allele frequencies of this polymorphism are significantly different between western commercial pig breeds Large white, landrace and Chinese native breeds Meishan.Association analysis between the polymorphism and fat deposition traits was performed in 234 Large white×Meishan F₂ pigs. Significant associations of the genotype with fat deposition traits such as leaf fat weight (LFW), internal fat rate (IFR), backfat thickness (BT), fat percentage (FP) and ratio of lean meat versus fat meat (RLF) were found. The individuals with GG genotype have lower fat deposition value than those with AA genotype and allele G was significantly associated with the decreasing of fat deposition.3 Real-time PCRUsing SYBR Green Real-time PCR, multiple different tissues including heart, liver, spleen, lung, kidney, stomach, intestine, ovary and fat were collected from a april aged Large white pig for gene expresstion. As a result, PTP-1B gene expressec in all eight tissues, it is expressed predominantly in lung, secondly in intestine and fat, very low in heart and kidney; For AQP7 gene, it is expressed predominantly in liver, secondly in heart, it is a little expressed in ovary and fat, hardly expressed in spleen and stomach.