The Cloning, Expression Pattern And Polymorphic Analysis On Four Genes From CAPN Family Of Porcine

Calpains, which have many physiological functions such as signal transduction, nervedevelopment, muscle growth, apoptosis and membrane fusion, are cytoplasmic cysteine proteasesrequiring calcium ions for activity. Calpains play an improtant role in muscle growth anddevelopment, myoblast fusion and differentiation, and they are the basic enzymes that lead to thetenderization of meat by cleaving myofibrillar protein including titin, nebulin and desmin. Thisstudy was designed to clone the cDNA and partial genomic sequences of pig CAPN1, CAPN3,CAPN7 and CAPNIO genes based on the sequences of human, rat, mouse and cattle CAPN genesby comparative genomics and bioinformatics approaches; to investigate the expressioncharacteristic of these genes by RT-PCR; to detect the polymorphisms of them by DNA sequencing,PCR-SSCP and PCR-RFLP methods. The main results are as follows:1. The cDNA sequences of domestic pig and wild boar CAPN7 genes were cloned byhomologous cloning, in silico cloning and RACE methods. The results showed that coding regionof pig CAPN7 gene is composed of 2442 bp encoding 813 AA.2. The cDNA sequences of domestic pig and wild boar CAPNIO genes were cloned by insilico cloning and RACE methods and two transcript variants were obtained. The results showedthat coding region of pig CAPNIO transcript variant 1 is composed of 2004 bp encoding 667 AA,while pig CAPNIO transcript variant 2 is composed of 1923 bp encoding 640 AA.3. The partial genomic sequences of pig CAPN1, CAPN3 and CAPN7 genes were cloned.4. The expression characteristic of CAPN1 gene was investigated by RT-PCR. The resultsshowed that the CAPN1 gene expressed in stomach, kidney, spleen, lung, heart, liver, large intestine,small intestine and uterus of Yorkshire at the age of birth, three-month, six-month, nine-month andtwelve-month; expressed in all detected tissues from Wild boar. There is no significant differencebetween thigh muscle and longissimus at the same growing period. The expression level inlongissimus of Yorkshire at nine-month and twelve-month are significantly less than that from birthto six-month.5. The expression characteristic of CAPN7 gene was investigated by RT-PCR. The resultsshowed that the CAPN7 gene expressed in stomach, kidney, spleen, lung, heart, liver, large intestine,small intestine and uterus of Yorkshire at the age of birth, three-month, six-month, nine-month andtwelve-month; expressed in all detected tissues from Wild boar. There is no significant difference between thigh muscle and longissimus at the same growing period. The expression level was themost in longissimus of Yorkshire at twelve-month.6. The expression characteristic of CAPN10 gene was investigated by RT-PCR. The resultsshowed that the CAPN10 gene expressed in stomach, kidney, spleen, lung, heart, liver, largeintestine, small intestine and uterus of Yorkshire at the age of birth, three-month, six-month,nine-month and twelve-month; expressed in all detected tissues from Wild boar. There is nosignificant difference between thigh muscle and longissimus at the same growing period. Theexpression level was the most in longissimus of Yorkshire at fifteen-day, nine-month andtwelve-month.7. The expression characteristic of CAPN3 gene was investigated by RT-PCR. The resultsshowed that the CAPN3 gene expressed only in stomach and muscle. There is no significantdifference between thigh muscle and longissimus at the same growing period. The expression levelwas the most in longissimus of Yorkshire at twenty-one-day, six-month and the least at seven-day.8. The expression characteristic of CAPN10 two transcript variants were investigated bycompetitive RT-PCR. The results showed that the expression level of transcript variant 1 washigher than that of transcript variant 2 in all tissues detected.9. 17 SNPs were found in CAPN1 gene by PCR-SSCP method, 12 of which were in exons andothers were in introns, and there were 4 missenses. Population genetics analyses showed that ineach polymorphic locus, there were significant difference among Yorkshire, Min pig and Wild boarin the distribution of genotylpes (P＜0.01), While no significant difference were found between Minpig and Wild boar except the locus detected by primer S1.10. 7 SNPs were found in 3' UTR of CAPN1 gene by DNA sequencing and PCR-RFLPmethod was developed based on restriction enzyme HinfⅠ/AlwNⅠto screen the population. Theresults showed that the distribution of genotypes among Min pig, Yorkshire, Duroc, Wild boar andCross bred were significant difference in the two loci (P<0.01 ).11. 4 SNPs were found in intron 7 of CAPN1 gene by DNA sequencing and PCR-RFLPmethod was developed based on restriction enzyme HinfⅠ, AlwNⅠand StuⅠto screen thepopulation. The results showed that at the NaⅢsite, the distribution of genotypes among Min pig,Yorkshire, Duroc, Wild boar and Cross bred were significant difference (P＜0.01), and threegenotypes were found only in Min pig at the StuⅠand HinfⅠsites.12. 1 SNP was found in intron 21 of CAPN1 gene by DNA sequencing and PCR-RFLPmethod was developed based on the restriction enzyme EcoRⅡto screen the population. Theresult showed that the distribution of genotypes among Min pig, Yorkshire, Duroc, Wild boar andCross bred were significant difference (P＜0.01).13. There were many differences in exon 1 of CAPN3 gene between Min pig and that fromGenBank. 2 SNPs were found in Min pig by PCR-SSCP method.14. PCR-RFLP analysis on the polymorphic locus leading to the change of HincⅡrecognition site in intron of CAPN3 gene showed that the distribution of genotypes were significant difference (P＜0.01) between Wild boar and other breeds, but no significant difference were foundamong Min pig, Yorkshire, Landrace and Duroc.15.3 SNPs in exon 4 of CAPN10 gene were found.