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Alternative TitleGenes essential to photoautotrophic growth of Synechocystis sp. PCC 6803
Thesis Advisor徐旭东
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword集胞藻pcc6803 光合自养生长 电子传递 果糖-1,6-/景天庚酮糖-1,7-二磷酸酶
Abstract集胞藻 (Synechocystis sp.) PCC6803是一种单细胞蓝藻,既可利用光能进行自养生长,也可利用葡萄糖进行异养生长,且具有天然的DNA转化系统,是光合作用分子生物学研究的重要模式之一。本研究以集胞藻PCC6803为材料,建立了随机插入诱变文库,并从中筛选出光合自养生长突变株,鉴定了与光合自养生长相关的一些基因,在此基础上,对基因ssr2060和slr2094在光合作用中的功能进行了研究。 主要研究内容和研究结果分3部分: 第一,光合自养生长突变株的筛选和基因定位。从两个集胞藻PCC 6803随机插入诱变文库的约11,000个克隆中筛选到28个与光合自养相关的突变株;利用反向PCR和测序进行的突变位点定位结果显示这些突变株涉及13个基因,其中1个是功能未知的基因,其余的12个则编码功能已知蛋白。 第二,对未知功能基因ssr2060的研究。通过对突变株的重构、下游两个基因ssr2061和slr1238的敲除以及互补实验证明ssr2060是光合自养生长必需基因。经预测,Ssr2060是跨膜蛋白,与其它氨基酸序列同源的蛋白在结构上有着高度的一致性。室温叶绿素荧光的分析发现突变株的电子传递受阻,利用各种不同的电子受体和电子供体研究光合电子传递链,发现受阻位点位于放氧复合体到QA之间;77K低温荧光光谱证明该基因的突变并不影响PSII的组装,但使突变株不能进行状态1的转变;不同光强下突变株的自养生长以及不同光质和不同光强下突变株的光合放氧证明该基因的突变并不影响光能的传递。 第三,对已知功能基因slr2094的研究。在集胞藻PCC6803中,slr2094编码双功能酶果糖-1,6-/景天庚酮糖-1,7-二磷酸酶。通过对突变株的重构证明基因slr2094是光合自养生长必需基因,突变株中没有果糖-1,6-二磷酸酶活性。不同光强下的混养生长和以luxAB作为报告基因检测基因在不同光强下的表达情况发现,突变株的混养生长只能在弱光下进行。 本研究得到以下主要结论: (1) ssr2060 (编码跨膜蛋白) 是集胞藻PCC6803的光合自养生长必需的基因;该基因突变后,放氧复合体到QA的电子传递受阻,不能进行状态1的转变,但PS II的组装和光能的传递不受影响。 (2) slr2094 (编码果糖-1,6-/景天庚酮糖-1,7-二磷酸酶) 是集胞藻PCC6803的光合自养生长必需的基因,并且突变株不能在强光下进行混养生长。
Other AbstractSynechocystis sp. PCC6803, a unicellular cyanobacterium, can utilize light energy to grow autotrophically or glucose to grow heterotrophically, and can be naturally transformed by exogenous DNA. It is one of the model species for the research of photosynthesis in cyanobacteria. In this study, photoautotrophic mutants were obtained by screening from random insertion mutants of Synechocystis sp. PCC6803, some genes that are potentially involved in photoautotrophy were identified, and the genes ssr2060 and slr2094 were further investigated for their roles in photoautotrophic growth. The main researches and results are in three parts: (1) Selection of mutants of photoautotrophic growth and identification of the mutated genes. Two random insertion mutant libraries of about 11,000 clones were constructed and screened, from which 28 mutants were selected that were incapable of growing photoautotrophically. By using inverse PCR, 13 genes were identified by sequencing the flanking DNA and searching the genome sequence of Synechocystis sp. PCC6803. Among these genes, ssr2060 is predicated to encode a function-unknown protein and the rest 12 genes encode known proteins. (2) The study of gene ssr2060 whose function is unknown. The reconstruction, targeted disruption of downstream ssr2061 and slr1238 and complementation experiments all showed that gene ssr2060 is essential for photoautotrophic growth of Synechocystis sp. PCC6803. Ssr2060 is predicated to be a transmembrane protein. The comparison of Ssr2060 with other cyanobacterial homologs showed that their structures are similar. Chl a fluorescence at room temperature suggested that the photosynthetic electron transport is defective in the mutant. By using the different donors and acceptors to check the flow of electrons through the photosynthetic electron transport, the inactivation of ssr2060 might block the electron flow from oxygen-evolving complex to QA. Fluorescence emission spectroscopy at 77K indicated that the mutation of gene ssr2060 is not involved in the assembly of PS II complex, while the conversion to state 1 is impaired. The mutant’s photoautotrophic growth at different light intensities, O2 evolution at different light wavelength and light intensities proved that energy transfer is not affected by the mutation of gene ssr2060. (3) The study of gene slr2094. The gene slr2094 encodes fructose-1, 6-/sedoheptulose-1,7-bisphosphatase (fbpI) in Synechocystis sp. PCC6803. The mutant was reconstructed and showed that gene slr2094 is indispensable for photoautotrophic growth. No enayme activity of fructose-1,6-bisphosphatase was detected in the mutant. The mixotrophic growth of mutant and the gene expression by using reporter gene luxAB at different light intensities showed that the mutant could grow mixtrophically only under low light intensity. The main conclusions of this study are: (1) ssr2060 (encoding a transmembrane protein) is an essential gene for photoautotrophic growth of Synechocystis sp. PCC6803. In the mutant, the electron transport from oxygen-evolving complex to QA is blocked and the conversion to state 1 is inhibited, while the mutation of ssr2060 doesn’t affect the assembly of PS II and the energy transfer. (2) slr2094 (encoding fructose-1, 6-/sedoheptulose-1,7-bisphosphatase (fbpI)) is essential to photoautotrophic growth of Synechocystis sp. PCC6803, and the mutant could not grow mixtrophically under high light intensity.
Document Type学位论文
Recommended Citation
GB/T 7714
阎春兰. 集胞藻PCC6803光合自养生长必需的基因[D]. 水生生物研究所. 中国科学院水生生物研究所,2006.
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