| Tibetan Pig (Sus scrofa, Tibetan pig) is indigenous to vast areas of Tibet, West of Sichuan, North of Gansu and Northernwest of Yunnan Province. It is the representative of the grazing breeds, which has unique resistance to harsh climate, poor feeding and veterinary preventation compared with other pig breeds. It has advantages of better resistance to stressful environment, more fine muscle fibre, more intramuscle fat and better flavour, and the disadvantages of low litter size, slow growth and low lean meat percentage. Due to its low productive performances, many exotic breeds have been introduced to cross with them sine 1950s. After 60 years' crossbreeding, the quantity of pure Tibetan Pigs is decreased dramatically. Nowadays, even in their provenance, it is difficult to find pure Tibetan Pigs. The breed is at risk of extinction. It is important to understand the rare breed resource before it becomes extinction.So far, a few papers have been reported about Tibetan pigs, and the researches related to resistance, meat quality and litter size candidate genes are even less. In addition, natural resistance-associated macrophage protein (NRAMP1), heart fatty-binding protein (H-FABP) and estrogen receptor (ESR) have been documented well in other breeds of swine. Herein, genes encoding above three proteins were chosen in Tibanten pigs in the present study.First of all, full cDNA of NRAMP1 was cloned with RT-PCR.The length of cDNA fragment is 1614 bp, encoding a peptide of 538 amino acid residues with a molecular weight of 58.8 Kda and isoelectric point 8.01. The predicted NRAMP1 protein is a typical NRAMP1, which is composed of a putative N-terminal proline- and serine-rich Src homology 3 (SH3)-binding domain, four phosphorylation sites for protein kinase C (PKC) , two N-linked glycosylation sites and twelve transmembrane (TM) domains. Sequence homology analysis showed that there are 13 nucleotides different between Tibetan pig and domestic pig but which is not resulted in the amino acid difference. By blasting the homologous sequences in GenBank database with demostic pig, dog, sheep, american bison red deer, horse, human and norway rat, their nucleotide sequence identities are 99.2%, 91.2, 88.7%, 88.1%, 86.8%, 85.8%, 83.2% and 71.6%, respectively.Secondly, NRAMP1 gene expression in 10 tissues of Tibetan Pig were analyzed by using semi-quantitative and real-time quantitative PCR methods. The gene expression was observed in all the tissues and no tissue-specific expression model was found in Tibetan Pig. Real-time quantitative PCR proved that the gene expression abundance was greatly different in various tissues. The maximal expression was observed in spleen and the minimum in liver. The average copy number is 3233 per ug cDNA. The difference between the maximal and the minimum is more than 77 times. The abundance of NRAMP1 expression in Tibetan Pig from high to low is in the following order: spleen, brain, lung, ileum, kidney, lymph node, colon, heart, muscle and liver. The finding suggests that there likely exists interspecies variation between Tibetan Pigs and demostic pig breeds.Thirdly, the genetic variation of H-FABP gene in 96 Tibetan Pigs were detected by PCR-RFLP with Mspâ… , Haeâ…¢, Hinfâ… . The results showed as follows: there are polymorphisms in Hinfâ… and Mspâ… locus, with the genotypes of HH, Hh, hh and AA, Aa, aa, among which the gene frequencies of H and A are 0.521 and 0.406. However, there is no variation in Haeâ…¢ locus, with the genotype of DD only.Last, the genetic variation of ESR gene in 96 Tibetan Pigs were detected by PCR-RFLP with Mspâ… , Haeâ…¢, Hinfâ… , meanwhile the relationship between ESR genotype and their litter size was analyzed. The results showed that there are 2 genotypes (AA and AB). The distribution of AA and AB is 0.75 and 0.25, respectively. The gene frequencies for A and B are 0.875 and 0.125, respectively. The litter size of AB type is 2.3 piglets more than that of AA.These findings are significant importance for realizing, preservation and utilization of the prescious resource in the future.
|
PDF Full Text Request