【作者】 程洁；

【导师】 包振民； 胡景杰；

【作者基本信息】 中国海洋大学 ， 遗传学， 2010， 博士

【摘要】 栉孔扇贝(Chlamys farreri)是我国北方最重要的海水经济养殖贝类之一,在海水养殖产业中占有极其重要的地位。而大片段基因组文库是当前分子遗传学和基因组学研究必不可少的工具,为了深入开展栉孔扇贝(Chlamys farreri)基因组学研究、阐明其基因组的结构与功能、图位克隆重要功能基因、构建高密度物理图谱并最终实现与已有遗传连锁图的整合,本研究成功构建了栉孔扇贝fosmid文库,并在其构建技术的基础上,以细菌人工染色体(Bacterial Artificial Chromosome,BAC)为载体,成功构建了栉孔扇贝的BAC文库。进一步通过栉孔扇贝fosmid文库和微卫星连锁图谱,对文库进行了基础研究的应用。主要结果如下：1、栉孔扇贝fosmid基因组文库的构建和鉴定本研究构建了第一个栉孔扇贝的fosmid基因组文库,该文库包含133,851个克隆。对随机挑取的80个克隆的限制性酶切分析表明,克隆的空载率低于1.25%,克隆的插入片段大小分布在30-45kb,插入片断平均长度为40kb。该文库覆盖栉孔扇贝基因组的4.3倍,得到任意单拷贝DNA序列的概率为98.7%。fosmid克隆的稳定性检测实验表明栉孔扇贝DNA在fosmid传代中表现得较为稳定,没有发现插入片段的丢失或重排。为了方便后续的PCR筛选,我们对所有的克隆进行了超级池和二级池的构建,并利用所构建的超级池和二级池筛选了2个基因和7个微卫星,结果阳性克隆数介于2到8之间。由此可见,该文库可以很好的用于目的基因或标记的筛选,将有利于物理作图和基因的图位克隆研究。2、栉孔扇贝BAC文库的构建和鉴定本研究以一只栉孔扇贝为材料,成功构建了两个栉孔扇贝BAC文库,分别为BamHⅠ文库和HindⅢ文库,其中BamHⅠ文库包含33,728个BAC克隆,平均插入片段约为80 kb,覆盖栉孔扇贝基因组约2.1倍；HindⅢ文库包含97,680个BAC克隆,平均插入片段约为102 kb,覆盖栉孔扇贝基因组约7.7倍。两个栉孔扇贝BAC文库总共由131,408个克隆组成,平均插入片段约为96kb,覆盖率为栉空扇贝单倍体基因组9.8倍。对随机挑取的60个BAC克隆进行限制性酶切分析表明,克隆的空载率低于1.67%,从文库中得到任意单拷贝DNA序列的概率为96.8%。BAC克隆的稳定性检测实验表明栉孔扇贝DNA在BAC传代中表现得较为稳定,没有发现插入片段的丢失或重排。为了方便后续的PCR筛选,我们对所有的克隆进行了超级池和二级池的构建。由此可见,该BAC文库可以很好的用于物理作图和基因的图位克隆研究,也为将来栉孔扇贝的大规模基因组测序提供了一个良好的平台。3、大片段基因组文库的应用：栉孔扇贝遗传图谱与物理图谱的整合初试本研究利用第一个栉孔扇贝fosmid文库以及微卫星遗传连锁图谱,通过构建fosmid文库的三维两步PCR筛选系统,选取分布于栉孔扇贝19个连锁群上的110个微卫星标记对部分文库(2.7×基因组)进行筛选和分析,总共102个微卫星标记(92.7%)经筛选得到至少一个阳性克隆,最终得到195个含有微卫星标记的fosmid克隆,每个标记的阳性克隆数介于1到7之间。这195个fosmid克隆与连锁图谱上的微卫星标记相关联,实现了fosmid克隆与遗传连锁图的整合。同时,我们构建了一个包含fosmid克隆、与其相关联的微卫星位点、扩增引物与PCR条件等相关信息的数据库。另外,本研究还将fosmid文库的三维两步PCR筛选与AFLP技术结合起来,初步尝试建立一种高通量的筛选文库中含有AFLP标记信息克隆的系统。我们用一对AFLP引物组合对fosmid文库的8个超级池进行了分析,共得到46个阳性克隆,这都为今后栉孔扇贝物理图谱的构建,以及高质量的遗传图谱与物理图谱的整合提供了一个经济、高效的技术平台。上述的研究,都是栉孔扇贝目的基因的筛选以及图位克隆,细胞遗传图和物理图的构建以及遗传连锁图谱和物理图谱整合等研究的有力工具,也为将来栉孔扇贝大规模基因组测序以及其结构和功能基因组学研究提供了良好的平台。更多还原

【Abstract】 Zhikong scallop(Chlamys farreri) is one of the most commercially important bivalves in China, but study on its genome is underdeveloped.To apply genome-based technologies for genetic improvements using marker-assisted selection, genome research involving large-insert genome library construction, genetic linkage mapping and physical mapping is required, and integration of genetic and physical maps would significantly enhance the capacities for genome research.In this study, the first fosmid library and another two BAC libraries of Zhikog scallop are constructed and characterized.Additionally, linkage-group specific clones from fosmid library are screened with markers on the microsatellite linkage map of Zhikong scallop. The major results are as follows:1.Construction and characterization of fosmid library in Zhikong scallopThe first Zhikong scallop fosmid library was constructed in this study. It consists of 133,851 clones with an average insert size of about 40 kb, amounting to 4.3 genome equivalents.Fosmid stability assays indicate that Zhikong scallop DNA was stable during propagation in the fosmid system. Library screening with two genes and seven microsatellite markers yielded between 2 and 8 positive clones, and none of those tested was absent from the library. The fosmid library will serve as a useful resource for physical mapping and positional cloning, and provide a better understanding of Zhikong scallop genome. 2. Construction and characterization of two BAC libraries in Zhikong scallopTwo Zhikong scallop BAC libraries were constructed with BamHⅠand HindⅢseparately, using only one scallop individual.The whole BAC library consists of 133,851 clones (33782 and 97680 for each) with an average insert size of about 96kb (80kb and 102kb for each),amounting to 9.8 X genome equivalents.BAC stability assays indicate that Zhikong scallop DNA was stable during propagation in the BAC system. The BAC library will serve as a useful resource for physical mapping and positional cloning, and provide a good tool for large scale sequencing of Zhikong scallop genome.3. Identification of linkage group specific clones from fosmid library of Zhikong scallop:A resource for integration of linkage and physical mapsThe first Zhikong scallop fosmid library was used to construct three-dimensional PCR screening system,110 microsatellite markers from 19 linkage groups of Zhikong scallop were used to screen a subset (2.7×)of the fosmid library. Of the 110 microsatellites,102 (92.7%) gave at least one positive clone and 8 (7.3%) failed to hit any clone. As a result,195 positive fosmid clones representing 19 linkage groups were identified, further verifying the genome coverage and utility of the library. These linkage group-specific clones provide resources essential for research of the Zhikong scallop’s genome, connecting linkage group to its chromosome, physical mapping and integration of the genetic map and physical map.Another, a high-throughput PCR-based screening method was developed which combines fosmid three-dimensional PCR screening system and amplified fragment length polymorphism (AFLP) technology.We used 1 AFLP primer to screen 8 superpools of the fosmid library,and got 46 AFLP-linked positive clones. This methodology allowed us to identify fosmid clones containing AFLP genetic markers,can link DNA-based physical map to the Zhikong scallop genetic map.This combination of approaches provides a low cost,efficient way to build high-quality integrated genetic and physical genome maps.更多还原