【作者】 魏兰珍；

【导师】 王全喜；

【作者基本信息】 华东师范大学 ， 植物学， 2010， 博士

【摘要】 蓝藻是一类能进行光合放氧作用的原核生物。集胞藻6803 (Synechocystis sp. strainPCC 6803)是一种单细胞蓝藻。该株系具有天然DNA转化系统,细胞结构简单,生长速度快,适应性强,是第一个完成基因组序列测定的光合生物。另外,它还能利用简单无机物合成有机物,并且表达的外源基因产物不易形成包含体。因此,利用转基因蓝藻制备基因工程药物的下游加工过程相对简化,是理想的基因工程研究的模式藻。但是,蓝藻中外源基因的表达效率低下,一直制约着蓝藻基因工程的长足发展。本研究以增强型绿色荧光蛋白基因egfp (enhanced green fluorescent protein)作为外源基因,构建适用于插入各种调控元件的同源重组整合平台——P0载体；然后利用该整合平台,通过插入各种已知的和未知的调控元件、自然转化、继代筛选,从而获得了多种转基因株系；通过比较各种转基因株系中外源基因的表达水平,对各种调控元件进行评价,从而筛选出能高效表达蓝藻外源基因的调控元件,具体步骤如下：(1)利用P0载体,在其报告基因上游分别插入聚球藻7942中groESL基因的温度诱导型启动子以及SD序列(GGAGAG),转化集胞藻6803,得到转基因Pg和Pg-s藻株。利用蛋白免疫印迹,研究不同生长时期、不同温度以及不同诱导时间下转基因株系中外源egfp基因的表达情况。研究结果表明,处于衰退期的转基因株系中外源基因的表达水平最高；并在此生长时期下,该启动子最适宜的诱导条件是在42℃诱导30分钟。通过比较各转基因藻中外源基因的表达情况发现：添加SD序列后则使外源基因的表达水平提高了1.6倍,而含groESL基因启动子和SD序列的Pg-s藻株中外源基因的表达水平比未添加启动子的P0藻株提高了2.0倍。综上所述,在集胞藻6803中,异源groESL基因启动子和SD序列的串联可明显提高蓝藻细胞中外源基因的表达水平。(2)利用P0载体,在报告基因上游插入聚球藻7002中cpcβ基因的光诱导型启动子,并转化集胞藻6803,获得转基因藻Pc。通过蛋白免疫印迹,研究不同光强、不同诱导时间以及不同CO2浓度对转基因株系中外源egfp基因的表达情况。结果表明：10μEm-2·s-1的光强诱导4小时后,转基因株系中外源基因的表达水平达到最高,而且低浓度的CO2也可以明显增强外源egfp基因的表达。(3)通过基因芯片数据和生物信息学的分析,初步确定ndhC-K-J操纵子可能的启动子区域,并插入到PO载体报告基因的上游区域,转化集胞藻6803,获得转基因株系P330。通过蛋白免疫印迹,研究不同生长时期、不同高光诱导时间以及二氧化碳浓度下转基因株系中外源基因的表达情况。结果表明,该未知启动子在高光诱导4小时,低二氧化碳培养条件下,使外源egfp基因的表达水平达到最高。通过比较Pg-s与P330藻株中外源基因的表达情况,揭示ndhC-K-J操纵子可能的启动子区域对外源基因的启动效果更为显著；P330藻株中外源基因的表达水平比Pg-s藻株提高4.2倍,比P0藻株提高8.2倍；蛋白定量结果表明,在转基因P330藻株中,外源egfp基因的表达量可达总蛋白的1.725%。本研究在构建同源重组双交换平台的基础上,分析来源于聚球藻7942的groESL基因启动子、聚球藻7002的cpcβ基因启动子、SD序列以及集胞藻6803自身ndhC-K-J操纵子可能的启动子区域对集胞藻6803中外源基因表达水平的影响。蛋白免疫印迹结果表明,这些调控元件均在一定程度提高了集胞藻6803中外源基因的表达效率,其中,ndhC-K-J操纵子可能的启动子区域对外源基因表达水平的提高幅度最大。以上结果为最终解决蓝藻细胞这一新型宿主系统中外源基因表达效率低下的问题注入了新的希望,为将来利用转基因蓝藻制备基因工程药物奠定了实验基础。更多还原

【Abstract】 Cyanobacteria are autotrophic prokaryotes performing oxygenic photosynthesis similar to that of higher plants. Synechocystis sp. strain PCC 6803 (Synechocystis 6803) is a unicellular cyanobacterium, and its complete genome is first characterized in phothosynthetic organisms. Moreover, this unicellular cyanobacterium possesses many characteristics, incuding the natural DNA transformation system, simple cell structure, fast growth, strong adaptability, and obtaining the useful organic products with little expenditure of energy and resources. Also, the products that exogenous genes expressed in cyanobacteria are not easy to generate the inclusion bodies, and most of cyanobacteria and their extract are non-toxic to the humans and animals. On the basis of these charactericts, cyanobacteria are found to be the perfect model algae for studing the genetic engineering. Although many expression plasmids of cyanobacterial cells have been extensively reported, the expression levels of foreign genes have not been optimized. Therefore, how to enhance exogenous gene expression levels in cyanobacterial cells remains unclear.The aims of this study were to enhance the expression levels of exogenous gene, enhanced green fluorescent protein (eGFP) gene, in Synechcosystis 6803 by changing different regulatory elements. These intresting findings are listed as follows:First of all, two homologous recombination vectors, P-groESL and P-groESL-SD, were constructed by inserting the groESL promoter gene, indued by temperature, originated from Synechococcus 7942, and the groESL promoter gene and SD sequence (i.e., GGAGAG) in the upstream of egfp, respectively. Subsequently, these 2 vectors were transferred to Synchocystis 6803 to correspondingly generate 2 transgenic algae, Pg and Pg-s. Further, the expression levels of exogenous gene, egfp, in Pg and Pg-s were investigated under different growth phases, temperatures, and time points by using immuoblotting. The results indicated that the optimal conditions for the expression of egfg in Synechocystis 6803 were at 42℃for 30 min. Under the optimal conditions, comparisons of the expression levels of egfp in Pg, Pg-s strains indicated that in addition to SD sequence in Pg-s enhanced 1.6 times than that Pg and the application of groESL promoter gene and the SD sequence in Pg-s enhanced 2.0 times than P0 strain. Taken together, these results indicated that the groESL-SD tandem was more efficient for the expression of egfp in Synechocystis 6803.Secondly, a homologous recombination vector, P0-cpcβ, was constructed by inserting the cpcβpromoter gene in the upstream of eGFP gene. Subsequently, the vector was transferred to Synechcysits 6803, by using nature transformation system, to generate the transgenic alga, Pc. Further, the expression levels of exogenous gene, egfp, in Pc were investigated under different light intensities, time points and CO2 concentrations by using immuoblotting. The results indicated that the optimal conditions for expressing the exogenous gene, egfp, were on exposure of Pc cells to the light of 10μEm-2·s-1 for 4 h with low CO2 concentration.Thirdly, on the basis of the gene chip data and bioinformatics analyses, the promoter region (330-bp) in the ndhC-K-J operon was determined preliminarily. Next, a homologous recombination vector, P0-330, was constructed by inserting the 330-bp predicted promoter in the upstream of eGFP gene, and then this vector was transferred into Synchocystis 6803 cells to generate transgenic alga, P330. Further, the expression levels of exogenous gene, egfp, in P330 were investigated under different growth phases, time points under high light, and CO2 concentrations by using immuoblotting. The results indicated that the maximum levels for the egfp expression were obtained under the exposure of P330 cells to high light and low CO2 for 4 h. Comparison of the expression levels of egfp in P330 and Pg-s (control), it was found that the expression amounts in P330 was approximately 4.2 times than that in Pg-s, and 8.2 times than that in P0; and attained 1.8% in total protein, indicting that a novel potential promoter, P330, is more efficient for expressing exogenous genes in cyanobacteria than other, identified promoters such as groESL, cpcβ.In this study, on the basis of the construction of the homologous recombination platform, the effects of many regulatory elements, including groESL promoter originated from Synechococcus 7942, cpcβpromoter originated from Synechococcus 7002, SD sequence, and novel potential promoter, P330, contained in the ndhC-K-J operon, on the expression levels of exogenous gene, egfp, were analyzed. Based on the results of immunoblotting, we found that all these regulatory elements can enhance the expression levels of exogenous gene in Synechocystis 6803 with different degrees. Of these regulatory elements, the predicted novel promoter in the ndhC-K-J operon is the most efficient for expressing the exogenous gene in cyanobacteria. These results will futhrer help in solving the low expression levels of exogenous gene in cyanobacterial host, and establishing the exprimental basis for obtaining efficiently the drugs by applying cyanobacterial genetic engineering.更多还原