【作者】 王景涛；

【导师】 柴宝峰；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 原生动物是区别于原核生物和高等真核生物的一个特定类群。它们作为一类单细胞真核生物，具有许多独特的特征，诸如，密码子使用的特殊性、单基因染色体结构和多核现象等，是分子细胞生物学研究的理想材料和模式生物。八肋游仆虫（Euplotes octocarinatus）是下毛目（Hypotrichida）游仆科（Euplotidae）的一个具有代表性的种。其具有一个无转录活性的生殖核（小核），负责有性生殖过程中遗传物质的传递；一个具转录活性的营养核（大核）负责细胞营养生长过程中基因的表达。下毛类纤毛虫的大核染色体被称为“基因大小染色体（gene-size chromosome）”，其含有一个基因编码序列，5′和3′端各有一段基因表达调控序列以及端粒序列（C₄）₄（A₄）₃，是真核生物中结构最简约染色体。这一特征是研究染色体结构和功能的一个优势，是本研究构建纤毛虫人工染色体，分析基因功能的基础。本研究首先克隆了八肋游仆虫的β₂-微管蛋白新基因（DQ864704），与β₁-微管蛋白基因相比，基因编码区（1335bp）的同源性达到99.1％。基因的非编码区（即上下游调控序列）的差异较大，但上游调控序列中均有一个TATAA盒，而且均富含AT。我们利用β₂-微管蛋白基因的上下游调控序列，构建纤毛虫大核人工染色体EUAMC，该染色体包括八肋游仆虫β₂-微管蛋白基因的上下游基因表达调控序列和端粒序列；动物绿色荧光质粒pEGFP-N₁（Clontech）的质粒元件-GFP基因、多克隆位点和抗生素基因（Kan^r）等。GFP基因将作为标记基因置于β₂-微管蛋白基因上下游调控序列之间。为了使GFP基因在八肋游仆虫中得到高效表达，用点突变的方法对该基因进行了密码子优化，便于原生动物细胞定位与基因功能的分析。用优化的脂质体转化方法将含有人工染色体的重组质粒pBTub-tel2转化到处于有性生殖分裂间期阶段的游仆虫细胞中，结果表明，GFP基因在游仆虫细胞中得以高效表达，36-48小时绿色荧光均匀分布在细胞核周围，逐渐扩散到整个细胞，染色体能够在大核中保持约90小时。更多还原

【Abstract】 Protozoa is a special and independent kingdom of microorganism in theprocessing of the evolution from prokaryote to eukaryotic cell. As aunicellular eukaryote, protozoa have many unique characters, such asdeviation from the universal genetic code, individual chromosomes andnuclear duality. It is perfect material and model biology for the research ofmolecular cell biology. Euplotes octocarinatus is a representational strain ofHypotrichida Euplotidae. It contains two types of nuclei in each cell, one isgenerative micronucleus, a typical diploid, meiotic germ-line nucleus, andanother one is vegetative macronucleus, a highly specialized expressionorganelle that provides all the transcripts for cell function.Hypotrichids ciliate macronucleus chromosome was named "genesize chromosome", which carrying the gene coding sequence flankedby short non-translated regions and bounded by telomeres （C₄）₄（A₄）₃.Macronucleus chromosome is furthest simple chromosome ineukaryotic cell. This character is benificial to investigating toconstruction and function of the chromosomes, and also toconstructing artificial chromosomes in ciliate and analyzing functionof gene.Firstly, we cloned a newβ₂-tubulin gene from the macronuclear andmicronuclear geneome of E. octocarinatus （DQ864704）, respectively.Compared withβ₁-tubulin gene, the coding regions of bothβ-tubulingenes have an identical length of 1335 bp （99.1％homology）. Thoughsequences of non-coding regions of two genes differ from each other,both of them contain a TATAA box in 5’ non-coding regions andaboundant in AT. Secondly, artificial chromosome EUAMC of ciliatewas constructed usingβ₂-tubulin non-translated regions of E.octocarinatus. The artificial chromosome was composed ofβ₂-tubulin non-translated regions, telomere, and plasmid elements of pEGFP-N₁ （GFPgene, multiple cloning site and Kan^r, etc）. Expression of GFP gene isregulated under the 5’ non-code regions ofβ₂-tubulin. For high expression ofGFP gene in E. octocarinatus cells, some codons in GFP gene wereoptimized by using point mutation to fit for the codon usage of ciliates.Plasmid pBTub-tel2, containing artificial chromosome, were transfectedinto Euplotes cells with the help of liposome. Interval observations under thefluorescent microscop were performed with excition waves of 470—490 nm.The results displayed that GFP genes were expressed effectively in E.octocarinatus, and GFP was distributed unifimly around macronuclearand diffused to the entire cell gradually. The chromosomes can be kept inthe macronucleus for 90h.更多还原