【作者】 武艳萍；

【导师】 王爱国； 李宁；

【作者基本信息】 中国农业大学 ， 动物遗传育种与繁殖学， 2005， 博士

【摘要】 转化生长因子β1(TGF-β1)是转化生长因子家族的重要成员,具有促进细胞生长、分化、迁移、免疫调节和细胞外间质(ECM)形成等作用。本研究以猪TGF-β1基因作为影响猪产仔数性状的候选基因,选用大白猪、长白猪、皮特兰猪、圣特西猪和二花脸猪为实验材料,利用单核苷酸构象多态性(single-strand conformation polymorphism,SSCP)方法对猪TGF-β1基因进行单核苷酸多态性的检测与分析,探讨其作为影响猪产仔数性状的候选基因的可能性。 首先利用本实验室构建的猪BAC文库TGF-β1阳性克隆测序获得第2外显子两端侧翼序列、第3外显子3’端、第5外显子3’端的侧翼序列,对TGF-β1基因cDNA序列和内含子4、内含子6进行了SSCP检测与分析,发现5个SNP:第四内含子32处(GenBank序列号:AF461809)C→T突变,第六内含子179处G→A突变、1043处C→T突变(GenBank序列号:AJ621785),和3’非翻译区2490和2494处G G→A A连锁突变(GenBank序列号:M23703)。将5个SNPs做单倍型处理后,群体中主要存在两种单倍型:CGCAA(记为A),和TATGG(记为K)。对不同猪种的单倍型频率统计发现,在大白猪、长白猪、皮特兰猪和圣特西猪中等引入猪种中,单倍型A为优势等位基因,在中国地方良种二花脸中,单倍型K为优势等位基因。建立线性模型:Y=u+场年季效应+TGF-β1单倍型构成效应+残差,估计各单倍型构成对大白猪产仔数的影响效应。结果显示以KK型的产仔数为最高。对三种单倍型构成的最小二乘均值进行两两t检验,KK型比AK型头胎总产仔数平均多产1.02头/胎,两者之间差异显著(P<0.05);KK型比从型多产0.49头/胎,但二者之间的差异不显著(P>0.1);从型比AK(型多产0.53头/胎,二者之间差异显著(P<0.1)。说明KK型对母猪头胎总产仔数有较大的影响。各单倍型构成对头胎产活仔数和二胎产仔数(包括总产和产活仔数)的影响差异不显著。对从型和AK型母猪断奶后第3天、妊娠第10天、15天、21天和70天的外周血血浆TGF-β1浓度检测结果显示:从型母猪断奶第3天的TGF-β1浓度显著高于妊娠第10、15、21和第70天(P<0.01),妊娠第10天的与妊娠第15天差异不显著,但显著高于妊娠第21天(P<0.01),妊娠第15天的TGF-β1浓度与妊娠第10天和第21天的差异都不显著。在另一种单倍型基因型AK型母猪中,也表现出类似的变化规律。从型和AK型母猪均以妊娠第70天的血浆TGF-β1浓度最低。AK型分别在5个采血日龄的血浆TGF-β1浓度均高于从型。对AA、AK(和KK型母猪妊娠第70天的TGF-β1浓度检测发现,KK型和AK型分别显著高于从型(P<0.01)。研究结果显示:TGF-β1基因对母猪产仔数有一定影响,单倍型构成KK是母猪产仔数优势基因型,杂合子AK型母猪产仔数最低。外周血TGF-β1浓度随母猪的生理周期而变化,不同单倍型构成在不同时期TGF-β1的表达量存在明显的差异,提示该因子在妊娠中具有重要作用。更多还原

【Abstract】 As a multifunctional cytokine, the transforming growth factor β1 (TGF-Bl) functions in epithelial cell proliferation, wound healing, immune response, angiogenesis and embryogenesis. In this experiment, We selected TGF-Bl as the candidate gene affecting litter size in pigs. We detected the polymorphisms of this gene by PCR-SSCP (single-strand conformation polymorphism ) in Large white (567), Landrace (174), Pietrain (39), Synthetic (56) and Erhualian(34), genetic correlation between the polymorphisms of TGF-Bl gene and litter size was analyzed.PCR-SSCP analysis was employed on a majority of TGF-β1 gene cDNA sequence, the intron 4 and the intron 6. The result showed that C→T mutation at 32nt (GenBank NO: AF461809) found in the intron 4 and G→A mutation at 179nt, C→T mutation at 1043nt (GenBank NO: AJ621785) found in the intron6, GG→AA linkage mutations at 2490nt and 2494nt respectively (GenBank NO: M23703) found in the 3’ flanking region. The haplotype analysis of five SNPs indicated that there are mainly two haplotypes of CGCAA(sign: A )and TATGG(sign: K ) in the experiment groups. Haplotype A is dominance in import breeds such as Large white, Landrace, Pietrain, and Synthetic, while haplotype K is dominance in Chinese native pig breed of Erhualian.Linear model was conducted for estimating the haplotypic combinations (HC) effects of KK, AK and AA. Least squares means for tatal number born (TNB) and number born alive (NBA) of the first and the second parities of KK were higher than that of AK and AA. By T test, the least squares mean for TNB of the first parity in KK was 1.02 piglets/litter higher than that of AK (P<0. 05), 0.49 piglets/litter higher than that of AA (P>0. 1). while the least squares mean for TNB of the first parity in AA was 0.53 piglets/litter higher than that of AK (P<0. 1). The result indicated that the KK genotype was significantly associated with litter size.Level of TGF-Bl in plasma of pregnant day 10,15, 21,70 and day 3 after weaning was assayed by the quantitative sandwich enzyme immunoassay technique. Individuals of HC AA and HC AK were investigated. For the AA genotype sows, TGF-Bl level at day 3 after weaning was much higher those at pregnancy. During pregnant period , level of TGF-Bl at day 10 was higher and declined gradually . Similarity occurred in AK genotype sows.The data further highlight TGF-β1 gene is associated with increased litter size.更多还原