【作者】 张伟；

【导师】 苏忠亮；

【作者基本信息】 青岛科技大学 ， 生物工程， 2011， 硕士

【摘要】 衣藻作为一种模式绿藻已应用于多种研究中。由于它具有多种优点:基因组序列已测序完成,遗传机制比较清楚;既能自养,又能异养;体内含有一个大的杯状叶绿体,容易被转化;生长周期短,光合效率高,因此素有“绿色酵母”之称,常用作宿主细胞来表达外源蛋白。衣藻是唯一一种在细胞核、叶绿体和线粒体中实现外源基因转化的生物。但外源基因在叶绿体中表达量低下一直是困扰衣藻叶绿体生物反应器商业化的瓶颈。影响外源基因表达量的最主要因素之一是启动子的强弱。强启动子能提高外源基因的转化频率,从而提高外源基因的表达量。衣藻叶绿体光系统II反应中心蛋白D1基因(PsbA)的启动子通常被认为是一种强启动子。本研究中,克隆了莱茵衣藻叶绿体PsbA启动子,以期PsbA启动子能提高外源基因在衣藻叶绿体中的转录效率。本研究的主要内容如下:首先,提取了莱茵衣藻DNA总基因组,设计PsbA启动子引物,并以总基因组为模板克隆了PsbA启动子。其次,构建了质粒p64D-PsbA-aadA。将PsbA启动子连接到质粒p64D中壮观霉素抗性基因(aadA)的上游。转化大肠杆菌DH5α,经氨苄青霉素和壮观霉素的筛选,获得了阳性单克隆斑。初步证明了PsbA启动子在大肠杆菌中能调控外源基因的表达。最后,构建了质粒psk-PsbA-gfp。将绿色荧光蛋白基因(gfp)连接到PsbA启动子的下游。转化大肠杆菌,经筛选后,获得了阳性单克隆斑。利用荧光显微镜,观察到了具有绿色荧光的大肠杆菌。证明了PsbA启动子具有启动活性。本研究成果为衣藻叶绿体基因工程的进一步研究奠定了基础。更多还原

【Abstract】 Chlamydomonas is used as a model green alga in many studies. Because it’s genome sequence has been sequenced, and the genetic mechanism is clear; it is a eukaryotic unicellular green algae; it is not only a autotrophic organism but also a heterotrophic organism; it contains a big cup chloroplast that can be easily transformed; it has short growth cycle and high photosynthetic efficiency, it is called“green yeaet”and usually is used as host cells to express foreign gene. The Chlamydomonas is the only organism that has been transformed successfully in nucleus, chloroplast and chondriosome. But the poor foreign gene expression level in chloroplast is a bottleneck that limits its using as bioreactor. The one of main factor affecting the foreign gene expression is promoter. The strong promoter can increase the transcription frequency and increase foreign genes expression level. The Chlamydomonas chloroplast endogenic photosystem II protein D1 promoter is usually considered as a strong promoter. In this research, the promoter above mentioned is cloned, and we hope that the promoter may be promote the foreign genes expesson in Chlamydomonas chloroplast.The contents of this study is as following:Firstly, the chlamydomonas total genome is extracted, and the PsbA primer is designed. With total DNA as templates, the PsbA promoter is cloned using the method of PCR.Secondly, the plasmid p64D-PsbA-aadA is constructed. The PsbA promoter is ligated to the spectinomycin resistance gene (aadA) upstream. The construction is transformed into E.coli DH5α. After screening on ampicillin and spectinomycin plate, the positive clone is obtained. Which proves that the PsbA promoter has the promoter activity.Finally, the plasmid psk-PsbA-gfp is constructed. The green fluorescent protein gene (gfp) is ligated to PsbA promoter downstream. After transforming E.coli DH5αand screening, the positive clone is obtained. Using the fluorescent microscope, the green fluorescence is observed in E.coli. This proves that PsbA promoter can regulate the green fluorescent protein gene expression in E.coli. The results of this research lay the foundation for further chlamydomonas chloroplast engineering study.更多还原