【作者】 鞠志花；

【导师】 安利国； 王长法；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2009， 硕士

【摘要】 κ-酪蛋白(以下简称κ-CN)通常是牛乳腺分泌的一种含有少量磷酸基的磷蛋白。它是牛乳中酪蛋白的组成之一,是凝乳酶的天然底物。在自然状态下,κ-CN是使牛奶保持稳定的乳浊液状态的重要因子。本文以600头中国荷斯坦奶牛为研究材料,以κ-CN基因为产奶性状的候选基因,采用DNA测序、PCR-RFLP和CRS-PCR技术,主要研究了κ-CN基因外显子4、外显子5和内含子4区基因序列的单核苷酸多态性与中国荷斯坦牛泌乳性状的关联性,以及第四和五外显子不同单倍型组合对泌乳性状的影响,为未来培育高乳蛋白及高乳脂率奶牛提供参考依据。研究结果如下:1.κ-CN基因Exon4多态性与泌乳性状关联性分析在exon4的第10891 bp、10927 bp、10988 bp和10996 bp处分别发生了T/C错义突变(导致136位异亮氨酸到苏氨酸的改变)、C/A错义突变(导致148位丙氨酸到天冬氨酸的改变)、G/A同义突变和T/A置换突变。据此分别选择了Taq?、HindШ、Pst?、SspI 4种限制性内切酶检测其多态性。4个位点的酶切多态性在所研究群体中是紧密连锁的;4个位点的A、B等位基因在群体中都有分布,且处于低度多态;A和B等位基因的频率分别为86.03%和13.97%;AA,AB和BB基因型频率分别为73.71%,24.63%和1.66%;χ2适合性检验表明,该群体在这4个位点的突变达到Hardy-Weinberg平衡状态(P>0.05);BB和AB基因型个体乳脂率显著高于AA基因型个体(P<0.05),AB基因型个体脂蛋白比显著高于AA基因型个体(P<0.05),但不同基因型对产奶量和乳蛋白率没有显著影响。2.κ-CN基因Exon5多态性和第四、五外显子单倍型组合与泌乳性状关联性分析在exon5上发现5个新的SNPs,包括A12907G、G12950A、C12989T、A13028G、T12980C,首次证实前4个位点紧密连锁。单位点基因型与泌乳性状的关联分析表明: T12980C突变位点的TC和CC基因型个体乳脂率、乳蛋白率分别极显著(P<0.01)、显著高于TT基因型个体(P<0.05);紧密连锁的4个突变位点的DE基因型个体乳脂率极显著高于DD基因型个体(P<0.01)。第四、五外显子9个多态位点单倍型组合分析表明:共构建出7种单倍型,发现了16种单倍型组合;其中H6H6单倍型组合个体乳脂率最高,H1H4次之;H1H4单倍型组合个体乳蛋白率最高;H5H6单倍型组合个体乳蛋白率及乳脂率最低。3.κ-CN基因Intron4多态性与泌乳性状关联性分析通过测序发现在intron 4基因序列存在3个新的SNPs,包括A12817G、G12218A、A12016G。与泌乳性状的相关性分析表明:A12817G 3种基因型间AG个体乳脂率极显著高于AA个体(P<0.01);G12218A突变位点的3种基因型间GA个体乳脂率和乳蛋白率均显著高于AA个体(P<0.05);A12016G突变位点不同的基因型对泌乳性状均无显著性差异(P>0.05)。更多还原

【Abstract】 Κappa-casein protein is phosphoprotein containing a small amount of phosphate that secreted by a cow mammary gland normally. It is a composition of casein in the milk and natural substrate of chymosin. In the raw state,κ-CN is an important factor to maintain a stable milk emulsion status.Theκ-casein gene was studied in 600 China Holsteins and regarded as a candidate gene for milk performance traits of cows. Sequencing、PCR-RFLP and CRS-PCR were applied to analyze the relationship between polymorphisms of 12 loci inκ–casein gene exon 4、5 and intron 4 and haplotype combinations of exon 4 and 5 in Chinese Holstein cattles and the milk performance traits for the purpose of providing molecular maker information to facilitate the breeding efficiency of high milk protein and milk fat. The results were as follows:1. Genetic polymorphisms ofκ–casein gene exon 4 and its correlation with milk performance traits in Chinese HolsteinsIn this study, the polymorphisms of four loci ofκ–casein gene exon4 were detected by PCR-RFLP and CRS-PCR with restriction endonuclease TaqI、HindШ、PstI、SspI. After sequencing, T/C、C/A、G/A and T/A SNP were identified at nucleotide 10891、10927、10988、10996 in exon4 ofκ–casein gene. The polymorphisms of the four loci in the experimental population is closely linked. Both alleles (A and B) of four loci were found in the population that showed low polymorphism. The gene frequencies of A and B were 86.03% and 13.97% separately. The genotype frequencies of AA, AB, BB were 73.71%, 24.63% and 1.66% respectively. Statistical results ofχ2 test indicated that four polymorphism sites in the population fitted with Hardy–Weinberg equilibrium (P > 0.05). Meanwhile, the effect of polymorphism ofκ–casein gene on milk production traits was analyzed, the results indicated that in the four loci, the cows with genotype BB and AB showed higher milk fat percent than those with genotypes AA(P < 0.05) ; with genotype AB showed higher fat protein ratio than those with genotypes AA(P < 0.05) ; the different genotypes ofκ–casein gene had no significant influence on milk yield and milk protein percent (P > 0.05).2. Genetic polymorphisms ofκ–casein gene exon 5 and haplotype combinations of exon 4 and 5 and its correlation with milk performance traits in Chinese HolsteinsFive SNPs in the exon five(A12907G、G12950A、C12989T、A13028G、T12980C) were found and A12907G、G12950A、C12989T、A13028G were closely linked. At locus T12980C, the cows with genotype TC and CC was very significantly (P < 0.01)and significantly higher than TT genotype, respectively (P < 0.05) ; with genotype TC and CC showed higher protein rate than those with genotypes TT(P < 0.05). At the remaining four loci, the cows with genotype DE showed higher fat rate than those with genotypes DD(P < 0.01). Haplotype combinations in theκ–casein gene exon four and five were studied. 7 haplotype and 16 haplotype combinations were found in 398 Chinese Holstein cattles, the cows with haplotype combinations H6H6 had the highest milk fat percentage and secondly was haplotype combinations H1H4. The cows with haplotype combinations H1H4 had the highest milk protein percentage and with haplotype combinations H5H6 had the lowest milk fat percentage and milk protein percentage.3. Genetic polymorphisms ofκ–casein gene intron4 and its correlation with milk performance traits in Chinese HolsteinsThree SNPs in the intron four(A12817G、G12218A、A12016G) were found by sequencing. The effect of polymorphism ofκ–casein gene on milk production traits was analyzed, the results indicated that in the A12817G loci, the cows with genotype AG showed higher fat rate than those with genotypes AA(P < 0.01); in the G12218A loci, the cows with genotype GA showed higher milk fat percent and protein rate than those with genotypes AA(P < 0.05); in the A12016G loci,the different genotype ofκ–casein gene had no significant influence on milk production traits (P > 0.05).更多还原