集胞藻PCC6803铜离子调控表达平台和新型插入突变方法的建立
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摘要
集胞藻(Synechocystis sp.) PCC6803是一种具有天然DNA转化系统的单细胞蓝藻,它既能利用光能进行自养生长,也可利用葡萄糖进行异养生长,是光合作用分子生物学研究的重要模式之一。本研究在集胞藻PCC6803中构建了一个铜离子诱导表达平台,利用这个平台对类囊体膜发生基因vipp1进行了研究;发展了一种新的基因插入突变方法,并对93个功能未知但在蓝藻和植物中高度保守的基因进行插入失活。
主要研究内容和研究结果分3部分:
(1)铜离子诱导表达平台的建立。克隆了集胞藻PCC6803的petE基因的启动子PpetE,并在集胞藻PCC6803中建立了一个受铜离子浓度诱导表达的平台,利用lacZ作为报告基因发现铜离子能调控lacZ基因的表达。
(2) vipp1基因的功能研究。利用铜离子诱导表达平台构建了一个vipp1基因的突变株(PpetE-vipp1),实验表明该突变株中vipp1受铜离子浓度的调控表达。我们的研究表明vipp1是细胞生命活动所必需的基因,它对于集胞藻PCC6803的光合放氧、色素含量、光合电子传递链以及类囊体膜结构的维持均起着重要作用。
(3)集胞藻PCC6803中未知功能保守基因的敲除。通过相似性搜索,发现在集胞藻PCC6803中存在93个与高等植物基因高度相似的未知功能基因。我们发展了一种基于T-A克隆的基因敲除方法并对这些基因进行了逐一敲除,发现56%的突变株中基因不能完全分离,表明这些基因可能是细胞生命活动所必需的。
本研究得到以下主要结论:
(1)铜离子诱导表达平台可以用于人为地调节基因在集胞藻PCC6803中的表达,是研究基因功能的良好工具。
(2) vipp1是集胞藻PCC6803生命活动所必需的基因,它对于细胞的光合放氧、色素的含量、光合电子传递链以及类囊体膜的结构的发生/维持均起着重要的作用。
(3)集胞藻PCC6803存在一些未知功能的保守基因,它们在蓝藻及高等植物中均非常保守,其中一些是细胞生命活动所必需的。
Synechocystis sp. PCC6803 is a unicellular cyanobacterium that is able to grow autotrophically using light energy or heterotrophically on glucose, can be naturally transformed by exogenous DNA. It is one of the research model species for genetic and molecular studies of cyanobacteria. In this study, a copper-induced gene expression platform was constructed in Synechocystis sp. PCC6803 and used to investigate a thylakoid membrane biogenesis gene vipp1, and a novel insertional mutagenesis method was developed and used to inactivate 93 genes of unknown functions but highly conserved among cyanobacteria and a higher plant.
The main researches and results are in three parts:
1) Construction of copper-induced gene expression platform in Synechocystis sp. PCC6803. The promoter of petE gene was cloned from Synechocystis sp. PCC6803 and a copper-induced gene expression platform was constructed using lacZ as the reporter gene. As seen with the result, the PpetE-driven expression of lacZ was regulated by the concentration of copper in the medium.
2) Functional studies of vipp1 gene in Synechocystis sp. PCC6803. A vipp1 mutant (PpetE-vipp1) was constructed on the basis of copper-induced expression platform, in which the expression of vipp1 was controlled by the copper. The results indicated that vipp1 was essential for the viability of Synechocystis PCC6803. It was probably involved in thylakoid membrane biogenesis or maintenance, and indirectly affected the oxygen evolution, photosynthetic pigments synthesis and photosynthetic electron transport in the cyanobacterium.
3) Insertion of genes of unknown functions and highly conserved. By similarity search, 93 genes of unknown functions were identified to be conserved among cyanobacteria and Arabidopsis. Insertional mutants of each of these genes were constructed. Of these mutants, 56% could not be completely segregated, suggesting that these genes might be essential for the survival of Synechocystis sp. PCC6803.
The main conclusions of this study are:
1) The copper induced gene expression platform is a useful tool for investigating the gene function in Synechocystis sp. PCC6803.
2) The gene vipp1 is essential for the viability of Synechocystis sp. PCC6803.
3) About half of the genes of unknown functions and highly conserved among cyanobacteria and plants are probably essential for the viability of Synechocystis sp. PCC6803.
主要研究内容和研究结果分3部分:
(1)铜离子诱导表达平台的建立。克隆了集胞藻PCC6803的petE基因的启动子PpetE,并在集胞藻PCC6803中建立了一个受铜离子浓度诱导表达的平台,利用lacZ作为报告基因发现铜离子能调控lacZ基因的表达。
(2) vipp1基因的功能研究。利用铜离子诱导表达平台构建了一个vipp1基因的突变株(PpetE-vipp1),实验表明该突变株中vipp1受铜离子浓度的调控表达。我们的研究表明vipp1是细胞生命活动所必需的基因,它对于集胞藻PCC6803的光合放氧、色素含量、光合电子传递链以及类囊体膜结构的维持均起着重要作用。
(3)集胞藻PCC6803中未知功能保守基因的敲除。通过相似性搜索,发现在集胞藻PCC6803中存在93个与高等植物基因高度相似的未知功能基因。我们发展了一种基于T-A克隆的基因敲除方法并对这些基因进行了逐一敲除,发现56%的突变株中基因不能完全分离,表明这些基因可能是细胞生命活动所必需的。
本研究得到以下主要结论:
(1)铜离子诱导表达平台可以用于人为地调节基因在集胞藻PCC6803中的表达,是研究基因功能的良好工具。
(2) vipp1是集胞藻PCC6803生命活动所必需的基因,它对于细胞的光合放氧、色素的含量、光合电子传递链以及类囊体膜的结构的发生/维持均起着重要的作用。
(3)集胞藻PCC6803存在一些未知功能的保守基因,它们在蓝藻及高等植物中均非常保守,其中一些是细胞生命活动所必需的。
Synechocystis sp. PCC6803 is a unicellular cyanobacterium that is able to grow autotrophically using light energy or heterotrophically on glucose, can be naturally transformed by exogenous DNA. It is one of the research model species for genetic and molecular studies of cyanobacteria. In this study, a copper-induced gene expression platform was constructed in Synechocystis sp. PCC6803 and used to investigate a thylakoid membrane biogenesis gene vipp1, and a novel insertional mutagenesis method was developed and used to inactivate 93 genes of unknown functions but highly conserved among cyanobacteria and a higher plant.
The main researches and results are in three parts:
1) Construction of copper-induced gene expression platform in Synechocystis sp. PCC6803. The promoter of petE gene was cloned from Synechocystis sp. PCC6803 and a copper-induced gene expression platform was constructed using lacZ as the reporter gene. As seen with the result, the PpetE-driven expression of lacZ was regulated by the concentration of copper in the medium.
2) Functional studies of vipp1 gene in Synechocystis sp. PCC6803. A vipp1 mutant (PpetE-vipp1) was constructed on the basis of copper-induced expression platform, in which the expression of vipp1 was controlled by the copper. The results indicated that vipp1 was essential for the viability of Synechocystis PCC6803. It was probably involved in thylakoid membrane biogenesis or maintenance, and indirectly affected the oxygen evolution, photosynthetic pigments synthesis and photosynthetic electron transport in the cyanobacterium.
3) Insertion of genes of unknown functions and highly conserved. By similarity search, 93 genes of unknown functions were identified to be conserved among cyanobacteria and Arabidopsis. Insertional mutants of each of these genes were constructed. Of these mutants, 56% could not be completely segregated, suggesting that these genes might be essential for the survival of Synechocystis sp. PCC6803.
The main conclusions of this study are:
1) The copper induced gene expression platform is a useful tool for investigating the gene function in Synechocystis sp. PCC6803.
2) The gene vipp1 is essential for the viability of Synechocystis sp. PCC6803.
3) About half of the genes of unknown functions and highly conserved among cyanobacteria and plants are probably essential for the viability of Synechocystis sp. PCC6803.
引文
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