【作者】 孙立彬；

【导师】 潘玉春；

【作者基本信息】 东北农业大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 钙蛋白酶系统在细胞内普遍存在，参与机体生长与代谢过程，尤其在肌原纤维更新和宰后肉嫩化中扮演重要角色。钙蛋白酶抑制蛋白（Calpastatin，CAST）是一种内源性的、需Ca2+激活的钙蛋白酶抑制剂，可抑制肌肉内蛋白质降解，降低肌细胞生长速度；屠宰后可抑制钙蛋白酶的活性，降低蛋白质水解。研究表明，猪CAST基因的产物参与肌肉生长过程中蛋白质的更新；在其它肉畜中则已发现，CAST基因的产物显著影响肉的嫩度。本研究利用PCR-SSCP、PCR-RFLP和直接测序方法对PIC商品猪和梅山猪、苏太猪、杜×长×大、长白×苏太、大白×苏太等分别进行CAST基因的多态性扫描和遗传效应研究，并对猪CAST基因部分序列进行了克隆及结构分析。具体工作及其结果如下：（1）猪CAST基因部分序列的克隆及结构分析：首次克隆了猪CAST基因的部分编码序列，并通过序列拼接获得了猪2型CAST基因的cDNA序列（Genbank acc. NO.AY555195）。通过对猪2型CAST基因进行结构预测及结构域分析，为进一步研究钙蛋白酶抑制蛋白在猪体内的功能打下了良好的基础。（2）猪CAST基因的多态性分析：分别利用PCR-SSCP、PCR-RFLP和直接测序方法来寻找多态位点。其一，对猪CAST基因的编码区用PCR-SSCP法扫描，结果所设计的引物的扩增序列均无多态。其二，对猪CAST基因利用PCR-RFLP法进行检测，结果在引物2-1和引物2-4分别发现单核苷酸多态位点。通过测序发现CAST基因在第200bp的位置（Acc. No. AY594692）发生突变，由A→G，导致HinfI酶切位点的消失；在第1187bp的位置（Acc. No. AY594692）发生突变，由C→T，导致MspI酶切位点的消失；在第998bp的位置（Acc. No. AY522920）发生突变，由A→G，导致HinfI酶切位点的消失；在第1189bp的位置（Acc. No. AY522920）发生突变，由C→T，导致TaqI酶切位点的消失。其三，通过直接测序结果发现，猪CAST基因第7外显子中发生突变，在第1448bp处由G→T，导致编码氨基酸发生变化，由Leu→Arg；第24外显子中发生突变，在第26bp处由T→C，导致编码氨基酸发生变化，由Ala→Val。这对我们继续研究猪CAST基因有重要的参考价值。（3）猪CAST基因的基因型及基因频率分布：2独立性检验统计结果表明，各基因型频率在不同猪群体间均差异极显著（P﹤0.01）。在各多态位点上，杜×长×大及苏太猪个体均表现为三种基因型，梅山猪个体表现为一种或两种基因型，大白×苏太和长白×苏太个体表现为两种或三种基因型。这可能是由于长期的自然选择和人工选择所造成的结果。（4）猪CAST基因SNPs与肉质性状和背膘厚的相关分析：对PIC125头杂交猪全部进行屠宰测定，采用PCR-RFLP方法和直接测序的方法找到CAST基因的多态性，并分析其在不同的基因型、饲料及基因型与饲料的互作效应对肉质性状和背膘厚这一重要的屠体性状的影响。结果如下：①不同的饲料对肌内脂肪、肌内蛋白、肉色有显著影响；②基因型效应对肌内脂肪含量和背膘厚具有显著影响；③基因型与饲料的互作效应对肌内脂<WP=10>肪、肌内蛋白、肉色有显著影响；④另外，性别效应对背膘的影响也是显著的。对梅山猪、苏太猪、杜×长×大、长白×苏太、大白×苏太等5个群体共110头猪全部进行屠宰测定，进一步探讨在相同的饲养水平下，猪CAST基因在不同的实验猪群体中其基因型效应对肉质性状和背膘厚这一重要的屠体性状的影响。结果如下：①基因型效应对嫩度和背膘厚具有显著影响（嫩度分别在A位点基因型间和B位点的基因型间差异显著（p<0.05）；而背膘厚在D位点基因型间差异显著（p<0.05））。②除pH24值和肉色在不同的实验群体间差异不显著（P﹥0.05）外，其余的测定指标在不同的实验群体间均差异极显著（p<0.01）；③肌内脂肪含量和肌内蛋白含量在猪的性别间均有显著差异（p<0.05）；综上，猪钙蛋白酶抑制蛋白基因可能是猪肉质性状和背膘这一重要的屠体性状的主效基因或与控制这些性状的主效基因连锁，并且本研究发现的DNA标记，能够用于猪的分子标记辅助选择。更多还原

【Abstract】 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<WP=12>×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更多还原