【作者】 汪星；

【导师】 赵开弘；

【作者基本信息】 华中科技大学 ， 环境工程， 2011， 博士

【摘要】 利用细菌双杂交技术,研究了在Synechocystis sp. PCC 6803基因组中与藻胆体核膜连接蛋白ApcE有相互作用的蛋白,结果发现糖基化转移酶S111466与ApcE有强相互作用。将sll1466在Synechocystis sp. PCC 6803体内敲除,并对所构建的突变体⊿sll1466进行了生理、代谢、遗传和光合作用等方面的研究,比较其与野生型(WT) Synechocystis sp. PCC 6803在上述功能方面的差异。研究结果表明：slll466在体内是单独转录的,与其他相邻基因没有共转录现象,sll1466的缺失不会引起藻内的其他极性效应；在缺乏sll1466的⊿sll1466中,ApcE的转录水平明显降低,使得藻胆体与光合作用中心的能量传递受阻,ApcE的表达量明显降低也使得⊿sll1466中藻胆体与类囊体膜的衔接不及WT中的紧密,从而导致⊿sll1466中藻胆体在类囊体膜上的移动速率较WT快；S111466的缺乏,也使得⊿sll1466中的聚β羟基丁酸酯和羧酶体的含量明显比WT中少,肝糖含量也不及WT,导致⊿sll1466的生长速率低于WT；sll1466的缺失使得藻对光的敏感性增强,在LL条件下,⊿sll1466对光的利用效率明显高于WT,其生长速率也较WT高,而⊿sll1466对HL的耐受能力则不及WT,生长速率也远不及WT,但我们发现Sll1466的缺乏并不影响藻的光状态转换和光系统的电子传递,只是⊿sll1466的状态转换速率要较WT快；在对藻的不同膜组分的糖基化检测中,我们发现通常在WT的细胞外膜和周质膜中起糖类转运作用的OprB孔蛋白Slr1908和S1r1841,在⊿sll1466中的类囊体膜上被检测出来,可见S1l1466对藻细胞内的糖类运输及代谢合成起着重要的作用；在对藻的不同膜组分的磷酸化检测中,作为藻胆体连接多肽的CpcG1蛋白在⊿sll1466的类囊体膜上清中被检测出来,而在WT的同样组分中未被检测到,可见⊿sll1466中的藻胆体更容易从类囊体膜上被洗脱下来；在对类囊体膜脂质的分析中,我们惊奇的发现,⊿sll1466中DGDG的含量较WT中有明显降低,该不饱和脂肪酸含量的显著降低,也是促使⊿sll1466中藻胆体与光合作用中心的能量传递受阻、对HL及高温的耐受能力不及WT、藻胆体在类囊体膜上的移动速率较WT快的重要因素；在缺乏S111466的条件下,藻对BG11中的一些离子和必要元素的需求量降低,其中以CO32-、Cu2+缺乏的培养条件尤为明显,从在缺乏CO32-的条件下⊿sll1466的生长速率高于WT我们推断⊿sll1466固定CO2的能力比WT强。在对Anabaena sp. PCC 7120的别藻蓝蛋白ApcE与ApcF的体内重组光谱及细菌双杂交验证两者相互作用的研究中,我们发现：在提纯前,PCB-ApcE/PCB-ApcF的吸收及荧光光谱峰与PCB-ApcF的峰位置一致,而提纯后,PCB-ApcE/PCB-ApcF的吸收及荧光光谱峰与PCB-ApcE的峰位置一致,表明ApcE与ApcF不能形成复合物。pBT-apcF与pTRG-apcE的共转化细胞能在无3-氨基-1,2,4三氮唑(3-AT)的非选择性培养基上的生长,而不能在有3-AT的选择性培养基上生长,说明在转化过程中未产生能抗3-AT的HIS3报告基因,进一步证实ApcE与ApcF间没有相互作用。在对Anabaena sp. PCC 7120藻胆体核亚基ApcD结合色素PCB的体内重组中,发现色素蛋白在提纯前后最大吸收和荧光峰发生了红移,从提纯前的605nm及633nm变为提纯后的650nm及665nm。为了研究该现象的原因,构建了ApcD的八个突变体,重组结果显示：突变体ApcD (Y88I)色素蛋白在提纯后的吸收和荧光光谱较提纯前均多出一个峰,分别为668nm和690nm；ApcD (W59Q)、ApcD (Y73A)、ApcD (W87E)色素蛋白在提纯前后的吸收和荧光光谱一致；ApcD (M126S)、ApcD(Y116S)、ApcD(M160T)色素蛋白在提纯前后的吸收光谱一致,而提纯后的荧光峰位置较提纯前分别红移了5nm、7nm和10nm；ApcD (M1151)色素蛋白在提纯前后的吸收和荧光光谱均发生了红移,从提纯前的605nm和633nm变为提纯后的638nm和655nm.这些色素蛋白在酸性尿素溶液变性条件下的最大吸收峰始终在662nm,表明辅基色素仍然是藻蓝胆素；在对PCB-ApcD、PCB-ApcD(Y116S)及PCB-ApcD(M160T)的圆二色谱分析发现,该两个氨基酸的突变均对脱辅基蛋白所连接的色素的构象产生了一定的影响,而对重组蛋白的二级结构没有影响。更多还原

【Abstract】 In Synechocystis sp. PCC 6803, a glycosyl transferase homologue Sll1466 was found to interact with the loop domain of phycobilisome core-membrane linker ApcE via BacterioMatchⅡTwo-Hybrid System.We focus on the physiology, metabolism, genetics and photosynthesis ofΔsll1466, in which the gene sll1466 was knocked out. The results show that:since no co-transcript with sll1466 was detected via the analyses of the transcription of the genes around sll1466, the mutation would not result in the polar effect. InΔsll1466 lacking S111466, the transcription of apcE decreased remarkably, and the integrality of PBSs was damaged, the PBSs inΔsll1466 moved more rapid, resulting in the inhibition of the energy transfer from phycobiliproteins to PSII; InΔsll1466, there was less amounts of the inclusions of carboxysomes, glycogen granules and poly-P-hydroxybutyrate than WT, indicating the less capacity of the metabolisms, which would also bring about in the lower growth; The shortage of sll1466 made the mutant more sensitive to light, and in LLΔsll1466 grew even better, while the mutant was less tolerant to HL, which would bring about in the lower growth; The loss of Sll1466 could not disable the state transition, the more rapid movement of the PBSs inΔsll1466 could be correlated to the more remarkable state transition and the more rapid relaxing rate back to the state transitionⅡ; In the isolation of the thylakoid membrane ofΔsll1466, the two OprB proteins Slr1841 and Slr1908 that are the carbohydrate-selective outer membrane porin, important to the transportation of carbohydrates, were detected to be glycosylated, indicating the importance of Sll1466 to the transportation of carbohydrates;InΔsll1466, CpcGl could be phosphorylated that was detected in the supernatant of thylakoid membrane, but in WT could not, which would upregulate the dismantling of PBSs; It was surprisingly discovered in the analysis of the lipid of thylakoid, DGDG decreased remarkably inΔsll1466. The variation of the contents of DGDG and related glycolipids would result in the phenotypes ofΔsll1466:the more rapid movement of PBSs, and the higher sensitivity to HL and temperature;Δsll1466 was more tolerant to the shortage of a series of essential ions in culture, e.g. the mutant grew better on the shortage of CO32- or Cu2+, implicating that it has the higher capacity of concentrating CO2.With the results of allophycocyanin proteins ApcE and ApcF from Anabaena sp. PCC7120 bound PCB in E. coli and two-hybrid bacteria system of ApcE and ApcF, we found that:theλmax of absorption and fluorescence spectra of PCB-ApcE/PCB-ApcF were the same as the Xmax of PCB-ApcF before purification, and the same as theλmax of PCB-ApcE after purification, indicating that ApcE and ApcF could not be complexed;The cells that transformed together with pBT-apcF and pTRG-apcE could grow in the non-selective growth medium, but could not grow in the selective growth medium containing 3-amino-1,2,4-triazole (3-AT), the HIS3 reporter gene was not transcribed and expressed, indicating that there were not interactions between ApcE and ApcF.Absorption and fluorescence spectra of the chromoprotein changed during the ApcD of phycobilisome core subunit from Anabaena sp. PCC 7120 bound PCB with reconstitution in E.coli, theλmax of absorption and fluorescence spectra were 605nm and 633nm before purification, while theλmax of absorption and fluorescence spectra were 650nm and 665nm after purification. To study the phenomenon above, eight mutants were constructed. It is indicated with reconstitution in E. coli that:the mutant ApcD (Y88I) had one more absorbance and fluorescence peaks after purification, theλmax of absorption and fluorescence spectra were 668 nm and 690 nm. The spectra of ApcD (W59Q), ApcD (Y73A), ApcD (W87E) had no change during the purification. The absorption spectra of ApcD (M126S), ApcD (Y116S), ApcD (M160T) had no change during the purification, but the fluorescence spectra had red shifts by 5nm,7nm, and lOnm after purification, respectively. Theλmax of absorption and fluorescence spectra of ApcD (M1151) had changed from 605nm and 633nm to 638nm and 655nm. Under acidic urea conditions, those chromoproteins had maximal absorption at 662nm, indicating that they had PCB chromophore. With the study of CD spectra of PCB-ApcD, PCB-ApcD (Y116S) and PCB-ApcD (M160T), it indicated that the mutations influenced the conformation of chromophore that bound with apoprotein, but did not affect the secondary structure of the reconstituted proteins.更多还原