【作者】 王晓亮；

【导师】 方盛国； 万秋红；

【作者基本信息】 浙江大学 ， 动物学， 2008， 硕士

【摘要】 扬子鳄(Alligator sinensis)是我国特有的珍稀濒危野生动物,长期以来,由于人类的捕杀和栖息地的丧失,其野外种群的数量已不足120条。为了保护这一珍稀濒危物种的基因资源,本论文以长兴扬子鳄自然繁育研究中心提供的血液样品为材料,以细菌人工染色体(BAC)为载体,建立了高容量的扬子鳄基因组BAC文库。同时在基因组BAC文库的基础上,利用PCR和DNA Walking步移法等技术,构建了扬子鳄MHCⅠ类基因组的克隆重叠群,从而为后续开展MHC基因组的物理图谱构建,以及MHC的基因组计划等工作,提供了关键的材料平台。主要研究结果如下:1.以新鲜血液为材料,利用pCC1BAC载体,成功构建了扬子鳄的基因组细菌人工染色体(BAC)文库。文库包含的164,352个BAC克隆,保存于1,712块96孔培养板上。经对随机挑取的150个BAC克隆进行脉冲电泳检测,计算出文库的平均插入片段为90kb,覆盖扬子鳄基因组约5.2倍(除去5.33%的空载体)。同时,从文库中获得任一单拷贝或多拷贝扬子鳄基因的概率为99.63%。2.用9对PCR引物筛BAC文库分别可以得到2-9个阳性克隆,平均每对能够在文库中成功扩增到4.78个阳性克隆,证实了所建文库具有较高的覆盖率。3.运用4D-PCR文库筛选技术,建立了扬子鳄基因组BAC文库的高效PCR筛选文库。该文库包括35个超级库和1190(35×34)个1D、2D、3D、4D超级亚库。任一引物或探针只需69个PCR反应,即可从BAC文库中筛选到所需的阳性BAC克隆。4.通过综合分析GeneBank数据库同源序列的特点,设计了筛库的兼并引物,通过优化引物最终得到了15个初筛阳性克隆,并利用DNA Walking步移法得到了3个初筛阳性克隆的侧翼序列,设计了3对MHCⅠ类基因的特异基因引物,对初筛克隆进行了进一步的确认,最终得到了4组BAC克隆重叠群:①.66B3、593C8和1542A12;②.298D4、423C6、453D5、500H12、1322A8和1715F3;③.678A8和751E7;④.20A2、202C7、280B2和941A9。更多还原

【Abstract】 The Chinese Alligator(Alligator sinensis)is rare and endangered species endemic to China.Due to environmental deterioration and habitat loss,the population of the Chinese Alligator declined rapidly.In order to protect the rare and endangered species,lots of the researches about Chinese Alligator conservation have begun.In this study,we constructed a high-quality bacterial artificial chromosome (BAC)library of the Chinese Alligator with fresh blood,which was provided by Changxing Research base of Chinese Alligator breeding.We also constructed the BAC contig of MHC class I gene of Chinese Alligator using PCR and DNA Walking method.It is very important to research the program of MHC,such as the constructing of the physical map of MHC.The main results are as follows:1.The Chinese Alligator bacterial artificial chromosome(BAC)library consisting of 164,352 clones has been constructed using fresh blood and pCC1BAC vector. The average insert size was calculated to be 90 kb based on the examination of 142 randomly selected clones,indicating that the Chinese Alligator library contained 5.2-fold genome equivalents.About 5.33%non-insert clones were observed in the 150 clones tested(8 out of 150).The probability of any Chinese Alligator gene being found in this library should be about 99.63%.2.Two or more positive clones(2-9)were found for these 9 loci,with an average of 4.78 positive clones per locus.The PCR screening results were thus in good agreement with an expected 5.2-fold genomic coverage of the library.3.Construction of high-effective rapid screening libraries by 4D-PCR.The 164,352 BAC clones were divided into 35 superpools and 1190(35×34) sub-superpools of 1D,2D,3D,4D.We can obtain the desired positive BAC clones by any primers or probes through 69 PCR reactions.4.We have designed the degenerate primer to screen the positive clones of Chinese Alligator MHC class I gene by the homologs counterpart of the MHC class I in the GeneBank database,and we finally got 15 positive BAC clones.We have designed further 3 Chinese Alligator MHC class I gene PCR primers based on 3 positive BAC clones’ flanking sequences,which have been gained by using DNA Walking method.By experiment and affirmance of aforementioned 3 PCR primers,we found 4 BAC contigs:①.66B3,593C8,and 1542A12;②.298D4, 423C6,453D5,500H12,1322A8,and 1715F3;③.678A8,and 751E7;④.20A2, 202C7,280B2,and 941A9.更多还原