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Alternative TitleBaiji’s genomic DNA library construction and application and the investigation on evolutionary correlation of mammalian C-value variation
Thesis Advisor王丁 ; 张先锋 ; 方盛国
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword白鱀豚 基因组文库 细菌人工染色体 Α乳白蛋白基因 图位克隆 基因组大小 C值 非编码dna 进化复杂度 生物多样性
Abstract得益于人类基因组计划(HGP)和其他模式生物基因组计划的顺利实施,生命科学研究领域正面临一个快速发展的契机。相对于其他哺乳动物在分子生物学研究领域的进展,鲸类动物在这方面的工作处于相对落后的局面。而鲸类动物是地球上体型变化最大、分布领域最广阔、在水生态系统食物链中位于顶级的一类哺乳动物,在进化上经历过独特的发展历程,因此具有明显区别于其他哺乳动物的遗传基础。开展鲸类基因组学研究将有助于揭示鲸类动物进化和适应的奥秘。本研究构建了白鱀豚Lipotes vexillifer的基因组文库,并对哺乳动物基因组进化进行了初步探讨。 本研究主要结果如下: 1) 以BAC载体pBeloBAC11为克隆系统,大肠杆菌DH10B为宿主菌,白鱀豚肺组织为基因组DNA来源,构建了白鱀豚的BAC基因组文库。 2) 对文库进行了鉴定。该文库共包含约有149,000个克隆,平均插入片段大小估计为83kb。 3) 用PCR的方法对文库进行了筛选。对白鱀豚的4个已知的核序列(α乳白蛋白基因,内光感受器类维生素A结合蛋白基因,短散布子Mago22座位侧翼区序列,和Mos (c-mos)基因的部分序列)进行筛选,分别得到了2,3,4,2个阳性克隆。结果显示,本研究构建的文库具有代表性。 4) 应用图位克隆技术得到了白鱀豚的α乳白蛋白全基因序列,证明本研究构建的基因组文库适合于白鱀豚的基因克隆。该基因全长1955bp,有4个外显子,通过和其他物种同源基因的比较发现这些外显子的大小、结构和序列具有高度保守性。 5) 应用CBCS法测定了一定量的BAC克隆的末端序列。从文库中随机挑选了20个BAC克隆,应用随机鸟枪法对BAC克隆的亚克隆直接测序,共得到了33条序列这些随机DNA序列将作为未来白鱀豚物理作图的路标。从而证明了本研究构建的白鱀豚基因组文库适合于从中筛选物理作图所需的随机DNA分子标记。 6) 本研究采用流式细胞术测定了白鱀豚和其他6种海洋哺乳动物,包括江豚Neophocaena phocaenoides、中华白海豚Sousa chinensis、白鲸Delphinapterus leucas、瓶鼻海豚Tursiops truncatus、加州海狮Zalophus californianus和斑海豹Phoca largha的C值。结果表明它们的C值分别为3.68pg,3.25pg,3.24pg,3.09pg,3.07pg,2.96pg和2.76pg;基因组大小分别为3.6×109bp,3.18×109bp, 3.18×109bp,3.02×109bp,3.00×109bp,2.88×109bp,2.68×109bp。 7) 应用本研究得到的7种海兽C值的数据,并结合目前已知的哺乳动物C值的数据,分析了哺乳动物C值变异和进化的关系。结果发现:哺乳动物的C值同其进化的结果(以生物多样性为代表)显著负相关,而同进化的过程(进化复杂度,以初始物种形成事件和辐射进化事件为代表)显著正相关。由于环境变化是进化过程中调整物种遗传物质的主要进化力,而非编码DNA的量有随环境变化不断增加的趋势,因此推测环境因子的稳定性可以对物种的基因组大小造成影响。
Other AbstractLife science is now facing an accelerated developmental moment profited from Human Genome Project and the analogous projects for model organisms. Compared to other placental mammals, cetacean researches are far droped behaind at molecular level, even cetaceans are the animals with the biggest changes of body-sizes, most world-wide distributions on the earth, and on the top niches in aquatic ecosystems. They have experienced a unique evolutionary process which is remarkably distinct to other mammals. In the present study, a BAC genomic DNA library for baiji, Lipotes vexillifer, was constructed, with some further preliminary investigations and discussions on genome evolution of placental mammals. The following goals were reached in this work: 1) Using pBeloBAC11 and E. coli DH10B as cloning system, and lung tissue as source of genomic DNA, we had constructed a genomic DNA library for baiji. Each step had been optimized so that they can be used as references for other cetacean’s library-construction. 2) The library consists of 149,000 clones with an average insert size of 83kb. Among 175 random selected BAC clones, 2 are pseudo-positive. Based on the estimated haploid genome size of 3.6×109bp for baiji, the library represents about 3.4 equivalents of its haploid genome. 3) PCR amplification for baiji’s four known nuclear genes, the alpha lactalbumin gene, the interphotoreceptor retinoid-binding protein (IRBP) gene, the SINE flanking sequence Mago22 locus and the Mos (c-mos) gene partial sequence showed that about 2, 4, 2, 3 positive clones were got from the 21 super pools, respectively. 4) By positional cloning we obtained the baiji’s alpha lactalbumin gene which showed that our library is well-suited for positional cloning other genes. The gene has a coding region of 1995bp with 4 exons. Compared to that of other eight mammal-species, the sizes, organization and sequences of exons are highly conserved. 5) 20 random selected BACs were end-sequenced by CBCS. We finally got 33 DNA segments among which there are 28 con-coding DNA, 2 single-exon genes and 3 internal exons of three different genes. The result showed that our library is suited for fishing out random DNA markers that can be used as landmark in future physical mapping. 6) The present study for the first time determined the C-values of seven marine mammal species including baiji, finless porpoise (Neophocaena phocaenoides), Chinese white dolphin (Sousa chinensis), Beluga whale (Delphinapterus leucas), bottlenose dolphin (Tursiops truncatus), Californian Sea lion (Zalophus californianus) and Largha seal (Phoca largha) by flow cytometry with chicken (Gallus domesticus) erythrocyte DNA content as standard. Their C-values are 3.68pg, 3.25pg, 3.24pg, 3.09pg, 3.07pg, 2.96pg and 2.76pg, respectively. Their genome sizes are 3.6×109bp, 3.18×109bp, 3.18×109bp, 3.02×109bp, 3.00×109bp, 2.88×109bp and 2.68×109bp, respectively. 7) Using the nowadays available C-value data for placental, we analyzed the evolutionary correlations of mammalian C-value variation. Results showed that mammals’ genome size variation negatively correlates with their evolutionary consequences presented by biodiversity and positively with their evolutionary process presented by numbers of incipient speciation and radiation events. For during evolution environmental changing is the major force that modulates organism’s genome, and the most majority of eukaryotic genome is non-coding DNA that increases following evolution, it’s assumed that stability of environmental factors can influence the genome DNA content.
Document Type学位论文
Recommended Citation
GB/T 7714
杜波. 白鱀豚基因组DNA文库构建及应用——兼论哺乳动物C值变异和进化的关系[D]. 水生生物研究所. 中国科学院水生生物研究所,2006.
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