扬子鳄基因组BAC文库及MHCⅡ类基因和OR基因物理图谱的构建
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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,由于环境恶化、栖息地丧失以及人类活动的影响,其野外种群的数量急剧下降,目前只有在安徽、浙江两省交界区域有所分布,种群数量已不足120条。为保护这一珍稀濒危物种的,我国政府已经采取了一系列措施。1972年将扬子鳄列为I级重点保护野生动物,1978年、1982年和1979年相继建立了安徽宣州扬子鳄繁殖研究中心、扬子鳄自然保护区、浙江长兴扬子鳄自然繁育研究中心等机构。
为了保护扬子鳄的基因资源,本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,成功建立了高质量的扬子鳄基因组BAC文库。以该文库为实验材料,构建了扬子鳄主要组织相容性复合体(MHC)II类基因和嗅觉受体(OR)基因的克隆重叠群,既验证了该文库的有效性,又对文库进行了基础研究的应用。其主要研究结果如下：
1.以新鲜外周血为材料,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216238个BAC克隆,保存于2254块96孔培养板上。经对随机挑取的150个BAC克隆进行电泳检测,计算出文库的平均插入片段为90kb,覆盖扬子鳄基因组约6.8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99.93%；
2.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46个超级库和1564(46×34)个1D、2D、3D、4D超级亚库。任一引物或探针只需80个PCR反应,即可从BAC文库中筛选到所需的阳性BAC克隆；
3.用7对扬子鳄特异性PCR引物进行文库筛选,平均每对能够在文库中成功扩增到6.1个阳性克隆,表明了所建文库具有较高的覆盖率,并与文库覆盖率计算结果(6.8)基本达成一致。
4.基于MHC II类以及OR基因的同源序列,成功设计了三对通用引物,并从扬子鳄基因组BAC文库中筛选到阳性BAC克隆的。
5.以这三对引物为基点,运用BAC质粒的末端测序和染色体步移,进行了一系列BAC文库阳性克隆的筛选,成功构建了两个克隆重叠群(contig)。
6.对OR基因克隆重叠群的9个BAC克隆进行测序,对所得数据进行分析,结果证明以上OR基因克隆重叠群的真实有效性,并对扬子鳄的OR基因进行了初步分析。
Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. For a long time, due to the environment deterioration, habitat loss and the influence of human activitiy, wild Chinese alligator population has dropped sharply to less than120. At present it appears restricted to southeastern Anhui Province of China, possibly very low numbers in Zhejiang and Jiangsu Provinces. To protect this species,in1972the Chinese Government listed Chinese alligator as a Class I endangered species. Anhui Research Center for Chinese Alligator Reproduction, Anhui National Nature Reserve for Chinese Alligator and Changxing Breeding and Research Center for Chinese Alligator were established in1978,1982,1979, separately.
In this study, for preserve the genetic resources, we constructed a high-quality bacterial artificial chromosome (BAC) library of the Chinese Alligator with fresh blood, which was provided by Changxing Breeding and Research Center of Chinese Alligator.Then further researchs show that this genomic BAC library is high quality and effective by constructing two contigs of MHC class II and OR genes of Chinese alligator. The main results are as follows:
1. The Chinese Alligator bacterial artificial chromosome (BAC) library consisting of216,238clones has been constructed using the whole blood and pCC1BAC vector. The average insert size was calculated to be90kb based on the examination of150randomly selected clones, indicating that the Chinese Alligator library contained6.8-fold genome equivalents. About5.53%non-insert clones were observed in the150clones tested (10out of174). The probability of any Chinese alligator gene being found in this library should be about99.93%.
2. Construction of high-effective rapid screening libraries by4D-PCR. The216,238BAC clones were divided into46superpools and1,564(46x34) sub-superpools of1D,2D,3D,4D. We can obtain the desired positive BAC clones by any primers or probes through80PCR reactions.
3. Using7Chinese Alligator speciese specific SSR PCR primers for screening the BAC library,6.1positive clones were got an average. The PCR screening results were thus in good agreement with an expected6.8-fold genomic coverage of the library.
4. We have designed two MHC class II PCR primers and one OR gene PCR primers by the homologous sequences.
5. Two contigs of Chinese Alligator MHC class II and OR genes were constructed by screening the genomic BAC library using the three primers in point4, BAC end sequencing and chromosome walking.
6. Though sequencing the nine BAC clones in the OR gene contig, we confirmed the high-quality of the BAC library. Then we conducted a preliminary analysis of OR genes in Chinese alligator genome.
为了保护扬子鳄的基因资源,本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,成功建立了高质量的扬子鳄基因组BAC文库。以该文库为实验材料,构建了扬子鳄主要组织相容性复合体(MHC)II类基因和嗅觉受体(OR)基因的克隆重叠群,既验证了该文库的有效性,又对文库进行了基础研究的应用。其主要研究结果如下：
1.以新鲜外周血为材料,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216238个BAC克隆,保存于2254块96孔培养板上。经对随机挑取的150个BAC克隆进行电泳检测,计算出文库的平均插入片段为90kb,覆盖扬子鳄基因组约6.8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99.93%；
2.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46个超级库和1564(46×34)个1D、2D、3D、4D超级亚库。任一引物或探针只需80个PCR反应,即可从BAC文库中筛选到所需的阳性BAC克隆；
3.用7对扬子鳄特异性PCR引物进行文库筛选,平均每对能够在文库中成功扩增到6.1个阳性克隆,表明了所建文库具有较高的覆盖率,并与文库覆盖率计算结果(6.8)基本达成一致。
4.基于MHC II类以及OR基因的同源序列,成功设计了三对通用引物,并从扬子鳄基因组BAC文库中筛选到阳性BAC克隆的。
5.以这三对引物为基点,运用BAC质粒的末端测序和染色体步移,进行了一系列BAC文库阳性克隆的筛选,成功构建了两个克隆重叠群(contig)。
6.对OR基因克隆重叠群的9个BAC克隆进行测序,对所得数据进行分析,结果证明以上OR基因克隆重叠群的真实有效性,并对扬子鳄的OR基因进行了初步分析。
Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. For a long time, due to the environment deterioration, habitat loss and the influence of human activitiy, wild Chinese alligator population has dropped sharply to less than120. At present it appears restricted to southeastern Anhui Province of China, possibly very low numbers in Zhejiang and Jiangsu Provinces. To protect this species,in1972the Chinese Government listed Chinese alligator as a Class I endangered species. Anhui Research Center for Chinese Alligator Reproduction, Anhui National Nature Reserve for Chinese Alligator and Changxing Breeding and Research Center for Chinese Alligator were established in1978,1982,1979, separately.
In this study, for preserve the genetic resources, we constructed a high-quality bacterial artificial chromosome (BAC) library of the Chinese Alligator with fresh blood, which was provided by Changxing Breeding and Research Center of Chinese Alligator.Then further researchs show that this genomic BAC library is high quality and effective by constructing two contigs of MHC class II and OR genes of Chinese alligator. The main results are as follows:
1. The Chinese Alligator bacterial artificial chromosome (BAC) library consisting of216,238clones has been constructed using the whole blood and pCC1BAC vector. The average insert size was calculated to be90kb based on the examination of150randomly selected clones, indicating that the Chinese Alligator library contained6.8-fold genome equivalents. About5.53%non-insert clones were observed in the150clones tested (10out of174). The probability of any Chinese alligator gene being found in this library should be about99.93%.
2. Construction of high-effective rapid screening libraries by4D-PCR. The216,238BAC clones were divided into46superpools and1,564(46x34) sub-superpools of1D,2D,3D,4D. We can obtain the desired positive BAC clones by any primers or probes through80PCR reactions.
3. Using7Chinese Alligator speciese specific SSR PCR primers for screening the BAC library,6.1positive clones were got an average. The PCR screening results were thus in good agreement with an expected6.8-fold genomic coverage of the library.
4. We have designed two MHC class II PCR primers and one OR gene PCR primers by the homologous sequences.
5. Two contigs of Chinese Alligator MHC class II and OR genes were constructed by screening the genomic BAC library using the three primers in point4, BAC end sequencing and chromosome walking.
6. Though sequencing the nine BAC clones in the OR gene contig, we confirmed the high-quality of the BAC library. Then we conducted a preliminary analysis of OR genes in Chinese alligator genome.
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