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摘要首先利用酶切和连接反应构建能使目的片段up500bp-mis16-hph-down500bp在大肠杆菌中扩增并得到单克隆菌落的重组质粒，再利用Mn2+驱动的mis16随机突变PCR技术建立mis16随机点突变文库，以重组质粒的形式保存。将文库质粒酶切以暴露mis16两端同源臂，根据双交换同源同组原理将目的片段转化入野生型单倍体粟酒裂殖酵母细胞中，以使突变mis16基因片段整合到酵母染色体中，通过观察酵母细胞转化子在限制温度36oC下的表型筛选出温敏突变型菌株，研究mis16具体突变位点，从而探究Mis16蛋白结构和功能之间的关系及其在Cnp1特异定位的分子途径中的作用。结果表明：随机点突变文库的建立是一个对Mn2+浓度不断摸索以找到最佳突变效果的过程，当Mn2+浓度为120 μM/mL和180 μM/mL时，mis16基因片段突变个数绝大多数为2~4个，且在位置上具有随机性，因而以这两种Mn2+浓度获得了理想的mis16随机点突变文库。19786
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关键词 核小体表观遗传 着丝粒 mis16 Mn2+ 易错PCR 随机点突变文库 温敏突变型
毕业论文设计说明书（论文）外文摘要
Title Point mutation of mis16 inducing cell lethality and identification of Mis16 functional domains
Abstract
Recombinant plasmids pBSK-up500bp-hph-down500bp were constructed via double digestion and ligation to expand the target gene up500bp-mis16-hph-down500bp and acquire monoclonal cell colonies after E.coli DH5α transformation. Error-prone PCR driven by Mn2+ was done to construct gene-specific random mutation library of mis16, which was stored in the form of recombinant plasmids. According to the principles of homologous recombination, the target gene was transformed into the wild-type haploid S.pombe cells, thus mutant mis16 genes were able to be integrated into the chromosomes of this kind of fission yeast. We screened temperature sensitive mutants through observing the phenotype of the yeast transformants plated at the restrictive temperature 36 oC to find the functional mutation sites, so that the relationship between protein structure and function of Mis16 and the roles it plays in Cnp1 deposition pathway will be clarified. The results show that the construction of gene-specific random mutation library of mis16 requires repetitive trials with many different Mn2+ concentrations. When the Mn2+ concentration come to 120μM/mL as well as 180μM/mL, the number of mutation sites which are very random is mostly 2~4. By using these two concentrations of Mn2+, we successfully constructed the gene-specific random mutation library of mis16.
Keywords nucleosome epigenetic inheritance centromere mis16 Mn2+
error-prone PCR random mutation library temperature sensitive mutants
目录
1 绪论 1
1.1 相关背景知识 1
1.1.1 裂殖酵母作为模式生物的优势 1
1.1.2 核小体表观遗传稳定性 2
1.1.3 着丝粒在细胞分裂中的重要作用 2
1.2 国内外研究现状与发展动态 3
1.2.1 与 DNA 复制相耦联 (replication-coupled)的核小体组装过程 3
1.2.2 PEV反映了染色质结构的动态变化 4
1.2.3 多种动粒蛋白调控着Cnp1正确运转 5
1.3 mis16在裂殖酵母基因组中的相关信息 6
1.4 研究意义及主要内容 7
2 材料与方法 8
2.1 材料及试剂 8
2.1.1 菌株 8
2.1.2 载体与引物 8