【作者】 徐阿娟；

【导师】 刘小林；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 本研究以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)。采用PCR-RFLP和CRS-RFLP方法,结合DNA测序技术检测了肿瘤坏死因子a(tumor necrosis factor-alpha, TNF-a)基因和S-腺苷高半胱氨酸水解酶编码基因(S-adenosylhomocysteine hydrolase encoding gene,AHCY)在荷斯坦奶牛群体中的多态性,并运用最小二乘分析模型对所检测到的多态位点与奶牛乳房炎的相关性进行了分析,以检验TNF-a和AHCY基因对奶牛乳房炎的遗传效应,以期发现影响奶牛乳房炎比较显著的遗传标记,为培育具有乳房炎抗性的奶牛品种打下基础,达到从根本上减少乳房炎的产生,减少乳房炎对奶业造成损失的目标。本研究共发现了10个多态位点(TNF-a基因的5′UTR的7个SNP,外显子区的2个SNP,GenBank收录号为GU129693;AHCY基因3′UTR的1个SNP),具体结果为:首先,TNF-a基因检测到第2外显子39 bp处的G→A的突变、第4外显子293 bp处C→T的突变、5′侧翼区PT3扩增区域的C→G的3个突变位点,这3个突变位点分别是DraI、AfaI和DdeI酶切多态位点,DraI为创造酶切位点。通过对突变位点的进一步分析,A、B等位基因在DraI酶切位点中的分布不均匀,而在AfaI和DdeI位点中属于均匀分布,但各位点的多态信息含量不一,DraI酶切位点的PIC<0.25,为低度多态,AfaI和DdeI位点0.25<PIC<0.5,这两个位点属于中度多态,且处于Hardy-Weinberg非平衡状态。三个位点的基因型和乳房炎显著相关,DraI、AfaI、DdeI位点相对应的AA、BB及AA易感乳房炎,BB、AA、AB对应于三个位点分别表现出乳房炎抗性。PT4位点发现A→C的突变(1 799bp处)、C→T的突变(1 851bp处)、C→T的突变(1 856bp处)、C→T的突变(1 872bp处),以及G→C和A→T的突变(分别位于1973bp和1 974bp处),在扩增区域所包含的内含子片段未发现多态位点。PT4位点的等位基因在感染牛群和健康牛群中分布不均匀(其数值与检测数值差别太大)。但各位点的多态信息含量较高,且处于Hardy-Weinberg非平衡状态。TNF-a基因PT4位点的11种基因型和奶牛体细胞评分效应之间的差异性较大。该位点的BB的SCSLAW值最大,HH基因型次之,AB的SCSLAW值最小,GG次之,则BB、HH基因型易感乳房炎,AB、GG基因型的抗性较强。其次,AHCY基因的外显子2、4、6、7、8、10及部分内含子区域均未发现多态位点,仅在3′侧翼区引物扩增区域的319bp处发现了T→G的突变,为Hin6I酶切位点。Hin6I酶切位点的A和B等位基因在感染牛群及健康牛群中的分布很均匀(有效等位基因数值接近于该群体所观测数值),处于中度多态,且处于Hardy-Weinberg非平衡状态。B在该位点中的感染牛和健康牛群中均属于优势基因,AA基因型的SCSLAW显著大于AB和BB基因型,BB基因型和奶牛乳房炎的抗性有关,Hin6I位点多态性和乳房炎表现出很强的相关性。更多还原

【Abstract】 Four hundred and seven Chinese Holstein cows were chosen and divided into 2 groups (controly group (317), sub-clinical and clinical group (100)) to investigate variations in bovine tumor necrosis factor-alpha (TNF-a) gene and S-adenosylhomocysteine hydrolase encoding gene and analyze their associations with mastitis. The aim of testing TNF-a and AHCY genes′genetic diversity and finding some genetic markers which associated with cow mastitis was to lay the root for develop dairy cattle breed which have the breast tissue resistant trait, and reduce the incidence of breast tissue and reduce its damage to the dairy industy. Ten polymorphic sites were detected (including 7 SNPs in 5′UTR and 2 SNPs in Exon2 of TNF-a gene, the embody number of GeneBank is GU129693; 1 SNP in 3′UTR of AHCY gene). The resμLts were showed as follows:G→A mutation at 39 bp in exon2 and one C→T mutation at 293 bp in exon4, and C→G mutation in 5′UTR were detected. The three polymorphic sites can be cut by DraI, AfaI and DdeI restriction enzymes respectively, the DraI locus was create restriction enzyme cutting Site. The further analysis of the mutant sites showed that the distribute of A、B allele frequency was uneven in DraI site, but it was uniform in AfaI and DdeI sites. In the three sites, the polymorphism information content (PIC) was different, it was low polymorphism in DraI site (PIC<0.25), it was moderate polymorphism in AfaI and DdeI sites (0.25<PIC<0.5), and the three sites were did not fit Hardy-Weinberg equilibrium. The three sites′genotypes were significantly related with cow mastitis. AA、BB and AA genotype has susceptibility to mastitis correspond to DraI, AfaI, DdeI sites, and BB, AA, AB genotype has resistibility to mastitis correspond to DraI, AfaI and DdeI sites. Six SNPs were detected in PT4 site, including the A→C mutation at 1799bp, C→T mutation at 1 851bp, C→T mutation at 1856bp, C→T mutation at 1 872bp G→C mutation at 1 973bp and A→T mutation at 1 974bp, there were no polymorphism in detected intron sites. The alleles in PT4 site were uniform in case and control cattle (effective number of alleles was far apart from the tested value). But the PIC was higher in every mutation site and not fit Hardy-Weinberg equilibrium in PT4 site. The effect of 11 genotypes on SCSLAW were different in PT4 site of TNF-a gene. BB genotype has the biggest SCSLAW, AB genotype has the smallest SCSLAW, BB genotype showed out the susceptibility to cow mastitis, AB genotype showed out the resistibility to cow mastitis.There were no polymorphism in Exon2、Exon4、Exon6、Exon7、Exon8、Exon10 and part intron region, there is only a T→G mutation site in 319bp of 3′UTR region, the polymorphic site can be cut by Hin6I restriction enzymes. The A and B alleles were uniform in AfaI and DdeI sites (effective number of alleles close to the tested value). It is moderate polymorphism in this site and did not fit Hardy-Weinberg equilibrium. B allele is the dominant gene in case and control cattle, AA genotype have a significant difference with AB and BB genotypes. BB genotype showed relevant to the resistance of mastitis, Hin6I site showed strong relevance.更多还原