【作者】 张洋；

【导师】 相建海； 张洪斌；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2008， 博士

【摘要】 基因组大片段BAC文库是进行生物遗传学和基因组学研究必不可少的基础工具。为了深入开展栉孔扇贝(Chlamys farreri)和凡纳滨对虾(Litopenaeus vannamei)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,本研究在植物基因组BAC文库构建技术的基础上,针对海洋生物的特点进行了大胆的改革与尝试,最终成功构建了C. ferrari和L. vannamei两种重要海水养殖动物的基因组BAC文库。本文构建的栉孔扇贝基因组BAC文库,由BamHI文库和MboI文库构成。其中BamHI文库含有73,728个BAC克隆,空载率约为1%,平均插入片段约为110 kb,覆盖栉孔扇贝单倍体基因组约8倍;MboI文库共有7,680个克隆组成,平均插入片段大小约为145 kb,插入率为100%,覆盖栉孔扇贝单倍体基因组约1.1倍。两个栉孔扇贝基因组BAC文库共由81,408个克隆组成,平均插入片段约为113 kb,覆盖率约为栉孔扇贝单倍体基因组大小的9.1倍。将栉孔扇贝基因组BAC文库的192个384微孔培养板中的73,728个BAC克隆以4 x 4点阵形式制备了高密度DNA薄膜,用于对感兴趣的基因及DNA序列的筛选。高密度DNA薄膜的覆盖率约为栉孔扇贝单倍体基因组的8.3倍。针对栉孔扇贝先天免疫系统通路的6个重要功能基因,根据栉孔扇贝cDNA序列以及异缘物种DNA序列设计了Overgo探针。利用Overgo探针对高密度DNA薄膜杂交筛选的结果显示,平均每个基因检测到7.3个潜在阳性克隆。本研究所构建的凡纳滨对虾基因组HindIII酶切BAC文库共有102,528个BAC克隆,存放于267个384微孔培养板中,平均插入片段大小约为101 kb,空载率约为5%,覆盖L. vannamei单倍体基因组约5倍。将其中240个384微孔培养板中的92,160个BAC克隆以4 x 4的矩阵排列形式制作了5张高密度凡纳滨对虾DNA薄膜,约覆盖整个对虾单倍体基因组的4.5倍。针对6个与对虾免疫、生殖生理有关的重要功能基因设计了Overgo探针,杂交筛选出20个阳性克隆,平均每个基因有3.3个潜在阳性克隆。以上筛选结果不仅为进一步研究这些功能基因的结构与功能、表达与调控,揭示它们在扇贝和对虾以及其他近缘种的免疫系统、抗逆和生殖生理过程中的作用机理打下了基础,同时也间接验证了栉孔扇贝和凡纳滨对虾基因组BAC文库将成为基因筛选、基因的结构与功能分析、基因图位克隆、物理图谱构建以及大规模全基因组测序等方面的有力工具。更多还原

【Abstract】 Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis and physical mapping of Zhikong Scallop, Chlamys farreri Jones et Preston and Pacific white shrimp, Litopenaeus vannamei, two BAC libraries were constructed from nuclear DNA of the two species, respectively. The scallop BAC libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99 % of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0 x haploid genome equivalents. Similarly, the MboI library consists of 7,680 clones, with an average insert size of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1 x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 7.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping and large-scale sequencing in the species.The shrimp BAC libraries were constructed in the HindIII site of the vectors pECBAC1 and pCLD04541, respectively. The combined libraries consists of 102,528 clones arrayed in 267 384-well microtiter plates, and approximately 95 % of the clones contain shrimp nuclear DNA inserts with an average size of 101 kb, covering 5.0 x haploid genome equivalents. High-density clone filters prepared from a subset of the pECBAC1 library were screened with Overgos designed from the cDNA sequences of six genes involved in immune system and sex determination related factor. Positive clones were identified for every gene, with an average of 3.3 BAC clones per gene probe. These results suggest that the shrimp BAC libraries provide useful tools for gene cloning, genome physical mapping and large-scale sequencing in the species.更多还原