【作者】 刘梅；

【导师】 王长法； 杨桂文；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2010， 硕士

【摘要】 疾病是影响畜牧生产的主要因素,在发达国家,由疾病造成的损失占畜牧生产总成本的17%左右,而在发展中国家,损失则高达35～50%。多种病原菌感染引发的奶牛乳腺炎,是奶牛常见的多发性疾病,给世界各国奶牛业造成巨大经济损失。目前已有多种方法来防控奶牛乳腺炎,虽然取得了一定的临床效果,但却引发了耐药菌株和抗生素残留等公共卫生问题。因而,应用现代分子生物学技术分析抗性和易感群体中候选基因的标记等位基因频率差异,结合遗传育种技术,从根本上提高家畜的抗病性与健康水平已成为当今家畜抗病育种研究的热点。动物的先天免疫反应通过模式识别受体(Pattern recognition receptors,PRR)特异识别病原微生物特定相关分子模式(Pathogen-associated molecular patterns,PAMP)来启动、触发。甘露糖结合凝集素(Mannose-binding lectin,MBL)就是一种广泛存在于人和动物体内的分泌型模式识别受体,能选择性识别多种细菌、病毒、真菌以及恶性细胞表面具有的甘露糖、N-乙酰甘露糖胺、N-乙酰葡糖胺等病原相关分子模式。通过MBL和其受体MBL相关的丝氨酸蛋白酶(MBL associated serine protease, MASP)结合,激活凝集素补体途径,发挥调理作用和中和作用,实现防御功能。在人中,发现MBL基因在启动子区和编码区存在6个单核苷酸多态(SNPs)位点,导致突变个体先天免疫功能紊乱,对多种病原易感性增强。因而我们推测MBL基因可作为奶牛乳腺炎的抗性候选基因。中国荷斯坦奶牛是由引进的纯种Holstein公牛与本地母牛的高代杂交种经长期选育而成,产奶量较高,但乳腺炎发病率也高达38%～50%。而鲁西黄牛和渤海黑牛是具有代表性的中国肉牛品种,耐粗饲、疾病感染率较低。本试验试图比较三个牛群中MBL基因的差异,寻找与奶牛乳腺炎抗性相关的分子标记。相关实验结果如下:采用巢式PCR、DNA测序和CRS-PCR(Created Restriction Site-PCR)方法,研究中国荷斯坦牛、鲁西黄牛、渤海黑牛的MBL1基因内含子1和外显子2的单核苷酸多态性,发现g.855G>A、g.2651G>A, g.2686T>C 3个SNPs,均为首次报道的位点,g.855G>A位于内含子1上,g.2651G>A和g.2686T>C位于外显子2上。其中g.2651G>A导致氨基酸的改变Val24Ile,g.2686T>C为同义突变。三个突变位点均已递交至NCBI, SNPs登录号分别为:ss172800380、ss172800389、ss172800394。MBL1基因g.855G>A、g.2651G>A和g.2686T>C 3个位点在中国荷斯坦牛、鲁西黄牛、渤海黑牛群体的优势等位基因相同,分别为G、G、C,其等位基因频率分别为0.87/0.58/0.57、1/0.75/0.74、1/0.76/0.63。经χ2适合性检验,荷斯坦牛在g.855G>A位点达到Hardy-Weinberg平衡状态(P>0.05),鲁西黄牛在g.855G>A、g.2651G>A位点达到Hardy-Weinberg平衡状态(P>0.05),渤海黑牛的所有位点均达到Hardy-Weinberg平衡状态(P>0.05)。3个品种牛在g.855G>A位点均表现为低度多态;在g.2651G>A和g.2686T>C位点均表现为中度多态(0.25<PIC<0.5)。对具有3个泌乳记录的596头中国荷斯坦牛MBL1基因进行研究,共构建出8种单倍型,发现19种单倍型组合。g.855G>A、g.2686T>C位点的突变与体细胞评分无相关性,但g.2651G>A位点与SCS具有显著相关性,揭示在乳腺炎抗性方面g.2651G>A位点可能起到重要作用。在试验牛群中,发现单倍型组合GGC/AAC个体的体细胞评分最低,具有乳腺炎抗性;单倍型组合AAT/AAT个体的乳蛋白率最高;单倍型组合AGC/AGC个体的305-d产奶量最高。因此,GGC/AAC, AAT/AAT和AGC/AGC三种单倍型组合可分别用来选育高乳腺炎抗性、高乳蛋白率及高产奶量奶牛的分子标记。分析了MBL1基因多态性与黄牛总补体活性的相关性,在国内初步建立了牛的总补体溶血活性测定方法,并对测定鲁西黄牛总补体活性的溶血试验条件进行了优化。更多还原

【Abstract】 Infectious disease has major adverse effects both on the economics of livestock production and on animal welfare. The costs of disease are estimated as 17% of turnover in the developed world (￡1.7 billion in the UK each year) and 35-50% in the developing world. Cow mastitis is a common disease in dairy industry worldwide, and has caused tremendous loss to the dairy production. A number of therapeutic, prophylactic and management strategies have been proposed to minimize this complex disease. Although these treatments have very good clinical outcome, emerge of drug-resistant strains and remaining antibiotics in milk have brought great concerns. Thus, an approach based on improving the host genetics in resistance to infectious diseases through molecular marker selective breeding is becoming widely accepted.Innate immune responses in animals are mediated by specific pattern recognition receptors or proteins that recognize microbe-specific pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide of Gram-negative bacteria, lipoteichoic acid of Gram-positive bacteria, and 1,3-β-D-glucan of eukaryotic fungi. This process also occurs in mammals by which secreted pattern recognition proteins such as mannose-binding lectin (MBL) and ficolins bind to microbial cells. Mannan-binding lectins (MBL) are collagenous C-type lectins involved in the innate immune response to various microbial pathogens. MBLs in plasma, like other collagenous lectins, are composed of multiple trimers, which further oligomerize toward the N-terminus by disulfide bridges and triple-helical association of the collagen-like domain to form higher order multimers. Antimicrobial functions of MBL, including opsonization, neutralization, and complement activation, depend on oligomeric assembly and clustered orientation of multiple carbohydrate-recognition domains which recognize repeating PAMPs on microbial surfaces. MBL selectively targets invading microorganisms for neutralization by its recognition domain’s binding to cell surface mannose and N-acetylglucosamine residues, then activating MBL-associated serine proteases (MASPs). In humans, the single-nucleotide polymorphisms (SNPs) in MBL gene were found to cause various innate immune dysfunctions.Chinese Holstein Cattle is derived from grading cross breeding and selection between the Chinese native cow and pure-bred bull of Holstein introduced to China. The frequency of mastitis in the breed is about 38%～50%. The Luxi yellow cattle and Bohai black cattle are two of the representative indigenous bovine (Bos taurus) breeds in China, which have been bred as beef and draft dual-purpose cattle for thousands of years because of the lower disease and enduring unfavourable feeding condition.In the present study we detected 3 single-nucleotide polymorphisms of MBL1 gene in Chinese native cattle and analyzed their associations with milk traits. By screening the genetic variation of MBL1 in 1053 individuals of three Chinese native cattle breeds including China Holstein, Luxi Yellow and Bohai Black using Created Restriction Site-Polymerase chain reaction (CRS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing techniques, three new SNPs were found. One SNP (g.855G>A) of MBL1 gene was located in intronⅠand the other two SNPs (g.2651G>A, g.2686T>C) were located in exonⅡ, which showed one non-synonymous mutation GTT (Val)>ATT (Ile) and one synonymous mutation GCT (Ala) >GCC (Ala). The SNPs were submitted to the National Centre for Biotechnology Information (submitter SNP numbers: ss172800380, ss172800389, ss172800394) In Holstein cattle and Luxi Yellow cattle and Bohai Black cattle, predominan allele were identical at g. 855G>A, g.2651G>A, and g.2686T>C, which were G, G, C and the allelic frequencies were 0.87/0.58/0.57, 1/0.75/0.74, 1/0.76/0.63, respectivly. Chi-square test indicated that g.855G>A of Chinese Holstein cattle, g.855G>A and g.2651G>A of Luxi Yellow cattle and all the sites of Bohai Black cattle were in accordance with the Hardy-Weinberg equilibrium (P>0.05). The value of polymorphism information content indicated that g.855G>A was slight polymorphism; g.2651G>A and g.2686T>C were moderate polymorphism in the three breed(s0.25<PIC<0.5). In 596 Chinese Holstein cattle with 3 lactation Dairy Herd Improvement (DHI) records, 8 different haplotypes and 19 genotype combinations were detected. Statistical analyses revealed no correlation between both g.855G>A and g.2686T>C and somatic cell score (SCS), however significant associations were found between g.2651G>A and SCS, suggesting a possible role of this SNP in the host response against mastitis. Our data also suggested that combined genotype GGC/AAC with the lowest SCS, AAT/AAT with the highest protein rate and AGC/AGC with the highest 305-d milk yield were favorable for the mastitis resistance and milk production traits. Thus, GGC/AAC, AAT/AAT and AGC/AGC can be used as a possible candidate for marker-assisted selection in dairy cattle breeding program.Optimum conditions for the total complement hemolytic activity assay in Luxi yellow cattle serum were also standardized.更多还原