Porcine Calpastatin Gene:Cloning, Polymorphism And Relationship With Meat Qualities And Backfat

Calpain system is ubiquitous in cells, involved in the course of growth and metabolizability of organism, especially in the renovating of the myofibril and postmortem tenderization of skeletal muscle. Calpastatin (CAST) is an endogenous inhibitor of the calpain system activated by Ca2+. It inhibits the decomposability of protein in muscle and reduces the growth speed of muscle cell. After slaughter, it can inhibit the activity of calpain and depressed the hydrolyzation of protein. Researches showed that the product of CAST gene participating in the renovation of protein during pork growth. And it has been found that CAST gene affect the meat tenderness in other livestock. In this research , we scanned the polymorphism on porcine CAST gene and studied the genetic effects in PIC commercial population and other five experiment populations(Meishan, Sutai, Duroc×Landrace×Yorkshire, Landrace×Sutai, Yorkshire×Sutai)with the methods including PCR-SSCP, PCR-RFLP and direct sequencing. Additionally, part sequence of CAST gene was cloned and sequenced. The works in detail and results were following: (1) Cloning and sequence analysis of part sequence of porcine CAST gene: it is the first time that part sequence of CAST gene of swine was cloned and sequenced in my research. Further, we assembled the complete coding sequence of porcine CAST typeⅡ(GenBank acc. NO. AY555195) and predicted the structure and domain of porcine CAST type Ⅱprotein with bioinformatics method , and this will make a good foundation for the following research on the function of CAST in swine. (2) Polymorphism detection of porcine CAST gene: we tried to find polymorphic sites by PCR-SSCP, PCR-RFLP and direct sequencing respectively. First, a scan to the coding region of porcine CAST gene by PCR-SSCP showed that no polymorphism was found in the sequences designed. Second, with primer 2-1 and primer 2-4, single nucleotide polymorphism (SNP) sites were found by PCR-RFLP on the CAST gene. It was found by direct sequencing that: there is a replace of nucleotide A by G in No. 200bp site, which resulted in the alter of HinfI site (Acc. No. AY594692); there is a transform from C to T in No. 1187bp site which resulted in the alter of MspI site (Acc. No. AY594692). A HinfI site disappears by the transform from A to G in No. 998bp site (Acc. No. AY522920); and a TaqI site disappears by the transform from C to T in No. 1189bp(Acc. No. AY522920). Third, direct sequencing showed that there is a mutation in the 1448th base of 7th exon ( G → T) in porcine CAST gene, which resulted in the alteration of amino acid encoding from Leu to Arg, and a mutation(T → C)in the 26th base of 24th exon resulted in alteration of Ala to Val. These results have important reference value for our further study in porcine CAST gene.(3) The frequency distribution of porcine CAST genotypes and alleles: 2 test showed the genotype frequencies are significant among different populations (P<0.01). To all of polymorphic sites, There are all three kind of genotypes in the population of Duroc×Landrace×Yorkshire and Sutai. But, only one or two kinds of genotypes in Meishan and two or three in Landrace×Sutai population and Yorkshire×Sutai population have been found in our research. This may be the result from long time nature selection and artificial selection.(4) We made slaughter test on all individuals of the first experiment group (125 PIC commercial pigs). After polymorphism was been detected with PCR-RFLP and direct sequencing, we analyzed the effects of nutrient level, genotype and the interaction between nutrient level and genotype to the meat quality traits and backfat thickness. The results were: ① the differences of nutrient level effects on the intramuscular fat (IMF) , intramuscular protein (IMP) and meat color are significant, but not on the backfat thickness; ② genotypes affect significantly on IMF and backfat thickness; ③ the interaction of genotype and nutrient level has significant effect on IMP, IMF and meat color, but no significant effect on backf...