【作者】 曹随忠；

【导师】 赵兴绪； 杜立新；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2005， 博士

【摘要】 乳房炎是造成奶牛业损失最严重的疾病之一,尽管在许多国家都广泛地实施了乳房炎防制措施,但它仍然是世界奶牛业面临的最大威胁。研究表明,奶牛乳房炎抗性在种间和种内都存在显著的遗传差异,通过遗传选择提高乳房炎抗性可能是解决这一重大疾病最好的长期策略。由于人们目前对控制奶牛乳房炎抗性的相关基因及其分子机制等还不清楚,这就严重阻碍了人们通过治疗、免疫接种和遗传选择等途径来显著提高奶牛对乳房炎的整体抗病力。本研究旨在通过分离和克隆与奶牛乳房炎抗性相关基因的探索性研究,初步揭示参与乳房炎抗性的基因种类和数量,为进一步深入开展乳房炎抗性的分子机理研究及将来通过遗传选择提高乳房炎的抗性提供理论基础。本研究取得的主要结果如下:1.应用抑制性消减杂交(SSH)技术成功构建了患乳房炎奶牛与健康奶牛外周血白细胞差异表达cDNA文库。以经产荷斯坦奶牛外周血白细胞为材料,分离Poly(A)+ RNA,反转录合成单链及双链cDNA,经酶切成为平均大小400~600bp的片段。将患乳房炎奶牛cDNA分成两组,分别与2种不同的接头连接,再与健康奶牛cDNA进行2次消减杂交和2次抑制性PCR扩增,将第2次PCR产物纯化后与pGM-T Easy载体连接,转化大肠杆菌TOP10感受态细胞进行文库扩增,最终构建得到了具有高消减效率的奶牛乳房炎抗性相关cDNA文库。文库扩增后得到610个白色阳性克隆,随机挑选16个克隆进行PCR鉴定,鉴定结果表明插入片段主要分布在250~750bp之间。2.应用反向Northern斑点杂交技术从消减cDNA文库中高通量筛选得到50个差异表达的克隆。分别用地高辛标记患乳房炎奶牛和健康奶牛cDNA作为杂交探针,对构建的cDNA消减文库利用反向Northern斑点杂交技术筛选了50个差异表达克隆,测序后得到48个质量合格的EST序列。根据在GenBank非冗余核酸数据库(nr)和EST数据库中BLASTn序列比对结果,将这些EST分为3类:第一类代表已知基因,共35个;第二类代表已知EST,共5个;第三类为新EST,共3个。代表已知基因的ESTs按照基因功能分为八类:信号转导与细胞间通信、细胞结构与运动、细胞凋亡、细胞与机体防御、物质转运、翻译与表达调控、代谢及其它。结合相关文献初步探讨了部分患乳房炎奶牛差异表达基因的作用和意义,其中WIP、bnbd5、AHCY、S100A12、MnSOD、SELL等基因可能在奶牛乳更多还原

【Abstract】 Bovine mastitis is a complex disease,which remains the most costly disease to the worldwide dairy industry, despite the widespread implementation of mastitis control strateges.Genetic differences of mastitis resistance between breeds and within breed in dairy cows are significant, so genetic selection to improve mastitis resistance may be the best long-term stratege bacause it offers the possibility for permanent resolution of this complex disease problem.Since the molecular mechanisms, gene systems and expression regulation of mastitis resistance in dairy cows remain unclear, it has seriously impeded our ability to substantially improve overall resistance to mastitis through therapies, vaccination programs and genetic selection schemes. The aim of the current research was to isolate and clone the mastitis resistant genes to look into molecular events associated with mastitis resistance and establish fundamental materials and techniques for further study and genetic selection. The main results obtained are listed bellow.To screen and clone mastitis resistant genes in dairy cows, differentially expressed cDNA library of peripheral blood leukocyte were constructed using suppression subtractive hybridization through following protocol: poly (A)+ RNA were purified using Oligotex mRNA Kits (Qiagen) from peripheral blood leukocytes of 20 Holstein cows with mastitis and 20 health Holstein cows in lactation as control; single- and double-stranded cDNA were synthesized from the poly(A)+ RNA using PCR-SelectTM cDNA Subtraction Kit (Clontech) and further digested using Rsa I into cDNA fragments sized from 400 to 600 bp (dscDNA), dscDNAs from the mastitis cows (as tester) were divided into two portions which were ligated separately with a different cDNA adaptor; the ligated cDNA were hybridized twice with the dscDNA of the healthy cows at 68℃for 8 h each; the products after double hybridizations were diluted by 200 times and then used for suppression PCR twice; the secondary PCR products was inserted into pGM-T Easy vector and transformed into E. coli TOP10 competent cells; 610 positive clones were obtained; identification of the inserted cDNA fragments in subtractive library was done using PCR. The results showed that there were inserted fragments of 250-750 bp in the 16 randomly selected positive clones, which would provide useful baseline for the screening and cloning of specific mastitis resistant genes and understanding the molecular mechanism of mastitis resistance in dairy cow.Differentially screening was performed using the reverse northern dot-blotting更多还原