奶牛乳铁蛋白基因5′侧翼区遗传多态性及其与乳腺炎关联性分析

牛乳铁蛋白(Lactoferrin, LF)是保护乳腺组织的防御因子之一, 是具有多种功能的糖蛋白。关于牛LF基因的多态性研究的报道较多, 但其多态性与奶牛乳腺炎相关性的研究较少。文章采用PCR-RFLP、CRS-PCR对268头中国荷斯坦牛LF基因启动子区的-926(G/A)、-915(T/G)、-478(/G)、+72(T/C)突变进行基因型分型, 应用最小二乘线性模型分析LF基因多态性与体细胞评分(Somatic cell score, SCS)的相关性。结果表明, 新发现+72(T/C)和-478(/G)对SCS有显著影响, 而其他两个位点对SCS影响不显著(P>0.05)。+72(T/C)的AB基因型是优良基因型, 其个体的SCS值均显著低于AA型(P<0.01)、BB型个体(P<0.05)。-478(/G)位点的C等位基因是优良的等位基因, CC基因型个体的SCS值极显著低于CD、DD基因型个体(P<0.01)。因此, LF基因+72(T/C)的AB基因型和-478(/G)位点的CC基因型均是奶牛乳腺炎抗性的优良基因型, 可作为分子标记应用于奶牛乳腺炎抗性筛选。     

奶牛乳铁蛋白基因5’侧翼区遗传多态性及其与乳腺炎关联性分析

牛乳铁蛋白(Lactoferrin,LF)是保护乳腺组织的防御因子之一,是具有多种功能的糖蛋白,关于牛LF基因的多态性研究的报道较多,但其多态性与奶牛乳腺炎相关性的研究较少,文章采用PCR-RFLP、CRS-PCR对268头中国荷斯坦牛LF基因启动子区的-926(G/A)、-915(T/G)、-478(/G)、+72(T/C)突变进行基因型分型,应用最小二乘线性模型分析LF基因多态性与体细胞评分(Somatic cell score,SCS)的相关性,结果表明,新发现+72(T/C)和-478(/G)对SCS有显著影响,而其他两个位点对SCS影响不显著(P>0.05),+72(T/C)的AB基因型是优良基因型,其个体的SCS值均显著低于AA型(P<0.01),BB型个体(P<0.05).-478(/G)位点的C等位基因是优良的等位基因,CC基因型个体的SCS值极显著低于CD、DD基因型个体(P<0.01).因此,LF基因+72(T/C)的AB基因型和-478(/G)位点的CC基因型均是奶牛乳腺炎抗性的优良基因型,可作为分子标记应用于奶牛乳腺炎抗性筛选.     

TNF-α基因多态性及其与奶牛乳房炎的相关性分析

以417头中国荷斯坦奶牛为研究对象,根据体细胞评分(Somatic cell score,SCS)的大小将该奶牛群体划分为感染牛群(100头)和健康牛群(317头)。通过PCR-RFLP和CRS-RFLP方法检测了肿瘤坏死因子α(Tumor necrosis factor-alpha,TNF-α)基因在荷斯坦奶牛群体中的多态性,并分析这些多态位点和奶牛乳房炎的相关性。研究发现了3个单核苷酸多态位点(Single-nucleotide polymorphism,SNP):第2外显子39bp处G→A的突变;第4外显子293bp处C→T的突变;5′侧翼区(5′-flanking region,5′UTR)C→G的突变。这3个突变位点分别是DraⅠ、AfaⅠ和DdeⅠ限制性内切酶的酶切多态位点,其中DraⅠ为创造酶切位点。经过基因型分析与χ2检验表明:3个酶切多态位点在荷斯坦奶牛群中均未达到Hardy-Weinberg平衡状态。运用SPSS13.0软件,采用最小二乘拟合线性模型分析3个酶切多态位点与SCS的关系,结果表明:AA基因型个体在DraⅠ酶切位点中的SCS显著大于BB及AB基因型个体(P0.05),BB基因型表现出乳房炎抗性。AfaⅠ酶切位点中BB基因型个体的SCS显著大于AA及AB基因型个体(P0.05),AA基因型表现出乳房炎抗性。DdeⅠ酶切位点中,AB基因型个体的SCS显著低于AA基因型个体(P0.05),AB基因型为优良基因型。因此BB、AA、AB基因型分别为DraⅠ、AfaⅠ、DdeⅠ酶切位点中的优良基因型,可作为分子标记应用于奶牛乳房炎抗性筛选。     

CXCR2基因多态性与奶牛乳房炎和乳品质的关联

采用PCR-SSCP技术研究了荷斯坦牛、西门塔尔牛和通江黄牛3个品种共160头个体CXCR2基因多态性与乳房炎抗性性状和对牛奶品质性状的遗传效应。结果表明: CXCR2基因有3个SNP多态位点, 分别位于序列的第685 bp、777 bp和861 bp位点, 确定了5个等位基因A、B、C、E和F。685 bp位点表现为BC和CC基因型, 777 bp位点表现为AA和AB基因型, 861 bp位点为CC和BC基因型。与奶牛乳房炎敏感性有关的基因型主要是BC、CC和FF, 可能对乳房炎有抗性的是AA、AB和EE基因型。据不同基因型对乳品质的遗传效应分析来看, AA、AB和EE基因型的乳品质性状极显著或显著优于其他基因型。     

奶牛HSP70基因3''-侧翼区多态性分析及其与生产性能的关系

以160头荷斯坦和娟姗奶牛作为研究对象,分别测定了其在不同温度条件下,直肠温度、呼吸频率、产奶量及乳成分,并进行了统计分析。同时设计引物扩增HSP70基因3’-侧翼区,通过PCR—SSCP技术分析hSPTO基因3’-侧翼区的多态性,并发现一个多态位点。结果表明：扩增片段为292bp,扩增产物有基因多态性,共发现4种基因型,分别为荷斯坦牛的AA（H）型、AB（H）型和娟姗牛的AA（J）刭、AB（J）型。其中B基因可能为抗热应激耩囚,这4种基囚型奶牛个体的生产性能差异不显著（P〉0．05）。     

荷斯坦牛Nramp1基因遗传多态性及其与乳房炎相关性的研究

利用PCR-SSCP技术检测了344头中国荷斯坦牛Nramp1基因exon 11的基因多态性, 并分析了其不同基因型与乳房炎及产奶量性状的关系。结果表明: 实验群体发现3种基因型AA、AB、BB, 其中A等位基因为优势等位基因, 等位基因频率为0.767, 而B等位基因频率则为0.233。经χ2适合性检验, 群体处于Hardy-Weinberg平衡状态(P>0.05)。测序结果显示: 扩增片段分别在200 bp(C/G)和254 bp(T/G)存在碱基突变, 并导致了氨基酸改变, 分别为丙氨酸替换为脯氨酸(Ala356Pro)、亮氨基酸替换为蛋氨酸(Leu374Met)。通过构建最小二乘线性模型, 进行Nramp1基因多态性与产奶量、体细胞评分(SCS)的相关性分析表明, AA型个体的SCS最小二乘均值显著低于BB﹑AB型(P<0.05), 而AA型﹑AB个体的产奶量最小二乘均值显著高于BB型(P<0.01, P<0.05), AA基因型可作为乳房炎抗性的优良基因型。因此, 可将Nramp1作为奶牛乳房炎候选基因应用于分子标记辅助选择育种。     

奶牛TLR4基因的多态性与乳房炎的相关性研究

TLR4通过识别病原体而激活免疫细胞,在先天免疫和适应性免疫防御中起着重要作用。以中国荷斯坦奶牛、三河牛和中国西门塔尔牛为研究对象,以TLR4为乳房炎抗性的候选基因,分别扩增316bp和382bp2个片段,分别采用SSCP和RFLP-AluⅠ方法来检测TLR4基因的多态性。结合测序发现:在intron1的第4,525bp处的A→G的突变,和exon3的第1,397bp处的T→C突变,使得产生多态。2个位点的A、B等位基因在3个群体中都有分布,且处于中度多态。χ2适合性检验表明,3个群体在这2个位点的突变达到Hardy-Weinberg平衡状态(P>0.05)。运用SAS8.0软件采用最小二乘法拟合线性模型,将基因座不同基因型与奶牛乳房炎进行了关联分析,结果表明:T4CRBR1的AA基因型为乳房炎抗性基因型(P<0.05),A等位基因为乳房炎抗性的有利基因,T4CRBR2的各基因型个体间的体细胞评分差异不显著(P>0.05)。     

中国荷斯坦牛IL8基因遗传多态性与泌乳性状以及体细胞评分的关联

以上海某奶牛场30个公牛家系的610头中国荷斯坦牛为试验材料,采用聚合酶链式反应-单链构象多态性(PCR-SSCP)技术对Interleukin-8(IL8)基因的遗传多态性进行了分析,采用混合动物模型分析了IL8基因突变位点与测定日产奶量、测定日乳脂率、测定日乳蛋白率、305d校正产奶量、305d乳脂量、305d乳蛋白量及测定日体细胞评分7个性状的相关性,寻找可用于生产实际的分子标记。共检测到KK、KA和AA3种基因型,频率分别为0.187、0.451和0.362,等位基因K和A的频率分别为0.412和0.588。该位点突变对测定日产奶量、305d乳蛋白量、305d校正产奶量和305d乳脂量以及体细胞评分影响达到极显著水平(P0.01),对测定日乳蛋白率的影响达到显著水平(P0.05),对测定日乳脂率影响不显著(P0.05)。多重比较表明:KK基因型对测定日产奶量、305d校正产奶量、305d乳蛋白量和305d乳脂量极显著高于AA和KA基因型(P0.01)。KK基因型的体细胞评分(SCS)最小二乘均值极显著低于KA、AA基因型(P0.01)。对于测定日的乳蛋白率AA基因型显著低于KA、KK型(P0.05)。IL8基因遗传突变对中国荷斯坦牛泌乳性状和乳房炎抗性有较大的遗传效应,可用于中国荷斯坦牛的分子标记辅助选择。     

奶牛β-Lg基因第1外显子PCR-SSCP多态性与泌乳性能的相关性

采用PCR-SSCP技术并结合测序对233头奶牛β乳球蛋白(β-Lg)基因5’端部分序列和外显子1全部序列进行了多态性研究,分析了该基因与奶牛泌乳性状的相关性。结果表明:β-Lg基因5’端和外显子1共存在2个等位基因3种基因型,BB型为优势基因型,B为优势等位基因。该群体在这一位点上偏离Hardy-Weinberg平衡状态,多态信息含量(PIC)为0.3548。测序结果显示,与普通牛该基因序列(X14710)相比,B等位基因在2073 bp、2202 bp和2206 bp处发生了G→C、C→T和A→G的碱基突变,其中2202 bp处的C→T突变导致第11位氨基酸由苏氨酸变为异亮氨酸,而A等位基因在3个位点上与X14710相同。最小二乘法分析表明,BB型305 d乳蛋白量显著高于AA型和AB型(P<0.05);AB型305 d乳脂量显著高于AA型(P<0.05),BB型与AB型之间差异不显著(P>0.05);等位基因B为高乳蛋白量和乳脂量的优势基因,可作为奶牛选育的分子遗传标记。     

κ中国荷斯坦奶牛κ-酪蛋白基因第四外显子多态性与产奶性状的关联分析

摘要: 以544头中国荷斯坦奶牛为研究对象, 以k-酪蛋白基因为产奶性状的候选基因, 扩增779 bp的片段, 结合测序结果采用PCR-RFLP方法来检测k-酪蛋白基因3个位点的多态性。结果在exon 4的第10 891 bp、10 927 bp和10 988 bp处分别发生了T/C、C/A错义突变和G/A同义突变, 据此分别选择了TaqⅠ、HindⅢ、 PstⅠ等 3种限制性内切酶检测了其多态性。发现3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%; c2适合性检验表明, 该群体在这3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; 3个位点的酶切多态性在所研究群体中是紧密连锁的。说明在中国荷斯坦奶牛群体中, κ-酪蛋白B等位基因可作为改良奶牛乳脂率性状的分子遗传标记。     

Novel SNPs in <Emphasis Type="Italic">HSP70A1A</Emphasis> gene and the association of polymorphisms with thermo tolerance traits and tissue specific expression in Chinese Holstein cattle

Heat stress induces heat shock proteins (HSPs) expression and HSP70 family is one of them that have been reported to involve in cellular protection against heat stress. But whether there is any association of genetic variation in the HSP70A1A gene with thermo tolerance is unknown. PCR-SSCP and DNA sequencing were used to detect possible SNPs in HSP70A1A gene in 890 Chinese Holstein cattle. Three fragments were amplified and five novel mutations were found in HSP70A1A gene in Chinese Holstein cattle. G/A mutation was found at nucleotide 1524 in coding region that resulting two genotypes of AA and AB. T/C mutation was found at nucleotide 3494 in 3′-UTR resulting three genotypes of CC, CD and DD. The other three point mutations, G/C at nucleotide 6400, C/T at nucleotide 6600 and G/A at nucleotide 6601 were also found in 3′-UTR resulting six genotypes of EE, EF, FF, EG, FG and GG. Linkage disequilibrium (LD) analysis showed that the nucleotide 6400, 6600 and 6601 were in strong LD (D > 0.75). Association analysis indicated that AB, DD and FF genotype were the thermo tolerance genotype. SBYE Green I was used to quantify HSP70A1A mRNA expression in different tissues through quantitative real-time PCR assay. The results of the real-time PCR showed that the expression of HSP70A1A mRNA in the heart was significantly higher than that in the other tissues (P < 0.05). We presume that these mutations could be used in marker assisted selection for anti-heat stress cows in our breeding program.     

Association of IL8 -105G/A with Mastitis Somatic Cell Score in Chinese Holstein Dairy Cows

The single nucleotide polymorphisms (SNPs) in the 5′ upstream of bovine IL8 gene were investigated in 810 Chinese Holstein cows from 35 bull families in a dairy farm in Shanghai using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. The Real-time PCR and Western blot were used to detect the mRNA and protein levels of genotype Chinese Holstein dairy cows. The results showed that one SNP -105G>A was detected, designating three genotypes (GG, GA and AA) with respective frequencies of 0.38, 0.46, and 0.16. The significant association of the SNP -105G>A with somatic cell score (SCS) was identified. Genotype GG had a significantly lower SCS than genotype GA or AA (P < 0.01), and the relative mRNA expression and protein level of GG was found to be the highest. These results suggest that the genotype GG may be a useful genetic marker for mastitis resistance selection and breeding in Chinese Holstein dairy cows.     

Novel SNPs of the Bovine PRLR Gene Associated with Milk Production Traits

The single nucleotide polymorphisms (SNPs) within exon 10 of the prolactin receptor gene (PRLR) were detected in Chinese Holstein cows using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods, and their genetic effects on milk production traits were evaluated in this study. Two newly detected SNPs (g.9206G→A and g.9681C→T) caused amino acid variations E378K and A536V, respectively, which were then preliminarily predicted at the topological level. Statistical results indicated that the two SNPs were significantly associated with milk yields, and cows with the combined genotype GGCC showed superior milk performance. A putative phosphorylation site was identified at residue 378K ([ST]-×-[RK]), which offers a partial explanation for the associations. These results suggest that the two novel SNPs within exon 10 of the PRLR gene associated with milk production traits are useful genetic markers in a selection program for Holstein dairy cattle.     

牛FSHR基因第10外显子单核苷酸多态性及其与双胎性状的关系

以秦川牛和荷斯坦奶牛的双胎母牛和单胎母牛为实验材料 ,以牛的FSHR基因的第 10个外显子作为标记牛双胎性状的候选基因 ,用SNP法进行了多态检测 .结果发现 ,在秦川牛的双胎母牛中突变率 6 0 % (6 10 ) ,而在单胎母牛中突变率为 2 0 % (2 10 ) ;在荷斯坦奶牛中 ,双胎母牛突变率为31 2 5 % (5 16 ) ,单胎母牛突变率为 6 6 7% (1 15 ) ;由此可见双胎牛和单胎牛二者之间FSHR基因的第 10个外显子的突变率差异明显 .这表明 ,选择FSHR基因的第 10个外显子有可能作为双胎性状的候选基因 .序列分析发现 ,在FSHR基因的第 15 0 6位碱基发生了突变 (T→C) ,但氨基酸没有发生变化 .     

中国荷斯坦牛白细胞黏附缺陷症PCR-RFLP检测方法的研究

本试验根据已知牛染色体上CD18编码基因序列设计引物,提取牛血液和精液DNA,可扩增出338bp的DNA片段,将PCR产物克隆到pMD18-T载体中,对阳性重组质粒进行测序,确定为牛的CD18基因。由于CD18基因的383位碱基由A变为G,而引起牛白细胞黏附缺陷症(BLAD),通过对济南市11个奶牛场356头奶牛及53头荷斯坦种公牛进行了BLAD的PCR-RFLP检测,共检出3头杂合母牛(携带者),占检测母牛群的0.84%,在荷斯坦公牛中只检测到一种基因型,没有发现隐性突变基因的携带者。     

Toll-Like Receptor 2 Gene Polymorphism and its Relationship with SCS in Dairy Cattle

Toll-like receptor 2 (TLR2) plays an important role in the innate immune response to a variety of pathogens. In this study, bovine TLR2 gene was taken as a candidate gene for mastitis resistance. Through PCR-SSCP analysis and sequencing, three missense mutations at T385 G, G398A, and G1884A were detected in the coding region that encoded extracellular domain. Altogether 240 dairy cattle of three breeds (Holstein, Simmental, and Sanhe cattle) were genotyped and allele frequencies were determined. The effects of TLR2 polymorphisms on somatic cell score (SCS) were analyzed and significant association was found between T385 G and SCS. The mean of genotype GG was significantly lower than those of genotype TT and TG. No significant associations were found with SCS for G398A and G1884A. Information provided in this research will be useful in further studies to determine the role of TLR2 gene in the mastitis resistance.