【作者】 康升云；

【导师】 王全喜；

【作者基本信息】 上海师范大学 ， 水生生物学， 2008， 硕士

【摘要】 自1968年蓝藻被证实具有遗传重组现象开始,越来越多的人致力于蓝藻基因工程研究。随着蓝藻基因工程的发展,有一批外源基因被导入到蓝藻中表达。主要涉及环境治理、代谢调控、营养保健品和基因工程药物表达等应用领域。但是,外源基因表达效率低下的瓶颈制约了蓝藻基因工程的发展,无法实现规模化生产应用。影响基因表达的因素有很多。如:基因拷贝数、密码子偏好性、启动子、终止子、反义RNA技术以及蛋白表达策略等。本研究试图从选择启动子、改变SD序列入手,对改进蓝藻外源基因表达系统和提高外源基因表达效率做一些探讨。集胞藻6803(Synechocystis sp. strain PCC6803)是一种单细胞蓝藻,具有生长速度快、培养条件简单、不产生毒素、细胞结构简单、遗传背景清楚、方便分子操作等的特点,在特殊培养条件下还可以进行异养生长,非常适合于利用光合生物反应器大规模生产。因此,集胞藻6803是种很好的蓝藻基因工程受体。本研究选择集胞藻6803染色体DNA中cupA基因及其下游相连的两个片段分别作为上游整合平台(up)和下游整合平台(down),在二者间插入诱导型高效启动子、egfp(增强型绿色荧光蛋白基因)报告基因、卡那霉素抗性筛选标记,构建得到集胞藻6803同源重组双交换整合平台。在整合平台的基础上,通过更换启动子和进行SD序列距离修饰,共得到6个带启动子和1个不带启动子的转化载体。把转化载体分别自然转化到野生型集胞藻6803中,经卡那霉素筛选得到7个转基因藻。它们分别含有能够被红光诱导的Pcpcβ(转pK-Pcpcβ集胞藻6803和转pK-Pcpcβ2集胞藻6803)、被温度诱导的PgroESL(转pK-PgroESL集胞藻6803和转pK-PgroESL2集胞藻6803)、被光照诱导的PpsbA(转pK-PpsbA集胞藻6803和转pK-PpsbA2集胞藻6803)和1个不带启动子的转pK-P0集胞藻6803。根据启动子的不同诱导特性,分别设计梯度诱导条件诱导EGFP表达,通过检测EGFP(增强型绿色荧光蛋白)荧光检测分析。EGFP在488nm光激发下会辐射绿色荧光,利用激光共聚焦显微镜对藻细胞直接进行荧光活性检测。通过比较EGFP的检测结果,判断各转基因藻外源基因表达系统的表达效率。本实验主要结果或结论:1.构建得到6个带启动子的转化载体;2.构建得到1个不带启动子的转化载体;3.通过自然转化获得了7个转基因藻株;4. PgroESL具有温度诱导增强表达作用;5. SD序列修饰具有上调表达作用。更多还原

【Abstract】 Since 1968 genetic recombinant phenomenon was found in cyanobacteria, more and more people committed to the cyanobacteria genetic engineering. With the development of cyanobacteria genetic engineering, many exogenous genes were introduced to cyanobacteria. It mainly involved environmental governance, metabolic, nutrition and health products, genetic engineering drugs,and so on. However, the inefficient expression for exogenous gene restricted the development and application of cyanobacteria genetic engineering.There are many factors affect gene expression in cyanobacteria. Such as, gene copy number, genetic code preference, promoter, terminator, antisense RNA technology and protein expression strategies. In this study, we choose different promoter and modify SD sequence, in order to improve the exogenous gene expression system of cyanobacteria and increase it’s expression efficiency.Synechocystis sp. strain PCC6803 is an unicellular cyanobacteria. It has faster growth rate and need simple culture condition. It’s cell structure is simple and genetic background is clear. It is facilitate operation and able to grow by either autotrophically or heterotrophically in special culture condition. It is an ideal biology to produce exogenous protein in large-scale bioreactor photosynthetic. Therefore, Synechocystis sp. strain PCC6803 is a very good receptor for cyanobacteria gene engineering.We choosed a fragment close with 3’terminal from cupA gene of Synechocystis sp. strain PCC6803 as the upstream integration platform(up) and another fragment linked to cupA gene as the downstream integration platform(down), and inserted efficient inducible promoter, egfp(enhanced green fluorescent protein gene), kanamycin resistance marker, constructed a homologous recombination double exchange integration platform.On the basis of the integration platform, we replaced the promoter and modified SD sequence, and gained six transformation vectors with promoter and one transformation vector with no promoter. The transformation vectors were transformed into the wild-type Synechocystis sp. strain PCC6803 cell. We gained seven transgenic cyanobacteria after the kanamycin screening. They contain the red-induced Pcpcβ(Transgenic pk-PcpcβSynechocystis PCC6803 and Transgenic pk-Pcpcβ2 Synechocystis PCC6803), the temperature-induced PgroESL(Transgenic pk-PgroESL Synechocystis PCC6803 and Transgenic pk-PgroESL2 Synechocystis PCC6803), the light-induced PpsbA(Transgenic pk-PpsbA Synechocystis PCC6803 and Transgenic pk-PpsbA2 Synechocystis PCC6803) and a non-promoter (Transgenic pk-P0 Synechocystis PCC6803).According to the promoter’s features, we designed gradient induced conditions to induce EGFP(enhanced green fluorescent protein) expression, and analysised expression efficiency by detecting EGFP fluorescence activity. Under the 488 nm excitation light, EGFP will eradiate green fluorescent. And the fluorescent from the active algal cells can be detected by using laser scanning confocal microscope directly. By comparing the test results of EGFP, we can judge the exogenous gene expression efficiency of the transgenic cyanobacteria.The main experimental results or conclusions including, 1)constructed six transformation vectors with promoter; 2)constructed one non-promoter transformation vector; 3)obtained seven transgenic cyanobacterium through natural transformation; 4)the PgroESL can increased the expression of EGFP after temperature-induced; 5)SD sequence modified can also up-regulated the expression of EGFP.更多还原