叶绿素缺失和异养生长对蓝藻Synechocystissp.PCC6803藻胆蛋白含量？…

通过遮黑培养缺失ｆｒｘＣ基因的蓝藻Ｓｙｎｅｃｈｏｃｙｓｔｉｓｓｐ．ＰＣＣ６８０３突变工程株,获得了叶绿素缺失的藻细胞,吸收光谱测定及数学计算表明,藻细胞中叶绿素缺失后藻胆蛋白含量增加,藻蓝蛋白和别藻蓝蛋白含量分别为相同条件下野生株对照组的４倍和６倍。野生株遮黑培养时,细胞进行异养生长,藻蛆蛋白含量下降,藻蓝蛋白和别藻蓝蛋白一分别为光照培养条件下自养生长的野生株细胞的３４．５％和２５．３％。另外,缺     

叶绿素缺失和异养生长对盐藻Synechocysitis Sp PCC6803藻胆蛋白含量影响

通过遮黑培养缺失frxC基因的蓝藻Synechocystis sp.PCC 6803突变工程株,获得了叶绿素缺失的藻细胞,吸收光谱测定及数学计算表明,藻细胞中叶绿素缺失后藻胆蛋白含量增加,藻蓝蛋白和别藻蓝蛋白含量分别为相同条件下野生株对照组的4倍和6倍。野生株遮黑培养时,细胞进行异养生长, 藻胆蛋白含量下降,藻蓝蛋白和别藻蓝蛋白含量分别为光照培养条件下自养生长的野生株细胞的34.5％和25.3％。另外,缺失apcE基因的突变工程株细胞的藻胆蛋白含量也少于对照野生株,表明apcE基础因的编码蛋白Lcm与藻胆蛋白的含量相关。     

蓝细菌Synechocystis sp.PCC6803中叶绿素合成调控类囊体膜重建的研究

利用ＤＮＡ体外重组技术构建了蓝细菌Ｓｙｎｅｃｈｏｃｙｓｔｉｓｓｐ．ＰＣＣ６８０３突变种ＯＲＦ４６９,它的染色体ＤＮＡ缺失ＯＲＦ４６９片段,该突变种在加入５ｍｍｏｌ／Ｌ葡萄糖的ＢＧ－１１培养基中经遮光培养２周后叶绿素全部消失,细胞甲醇提取液光谱中６６５ｎｍ处叶绿互峰消失,６２９ｎｍ处出现原叶绿素酸酯峰,完整细胞光谱仅存在６２０ｎｍ的藻胆蛋白肩峰,细胞的类囊体膜消失,但藻胆体颗粒并未减少;细胞培养物重     

集胞藻PCC6803中S2P同源蛋白基因sll0862缺失突变株对热胁迫与氧化胁迫的响应

原核生物中S2P参与应答外界环境刺激,然而行光合作用的蓝细菌-集胞藻PCC6803的S2P同源蛋白功能未知。【目的】考察集胞藻PCC6803中S2P同源蛋白sll0862是否参与外界环境刺激的应答。【方法】监测在高温和氧化胁迫的条件下sll0862基因缺失突变株与野生株在生长速率或存活率上的差异,利用水样调制叶绿素荧光仪(water-PAM,脉冲-振幅-调制叶绿素荧光仪)测量在高温和氧化胁迫的条件下突变株与野生株叶绿素荧光参数的差异,来考察其光合作用差异。【结果】sll0862突变株与野生株在正常的培养环境中生长速率并无差异,但是将sll0862突变株与野生株在48℃加热处理半小时后,sll0862突变株的存活率明显低于野生株。当初始OD730值为0.1的藻液中添加终浓度为1 mmol/L双氧水的时候,sll0862突变株的生长速率比野生株明显低,而且氧化胁迫条件下突变株与野生株的调制叶绿素荧光有差异。【结论】集胞藻PCC6803中sll0862基因的缺失导致突变体对高温与氧化胁迫响应出现缺陷,提示有功能的sll0862参与响应热和氧化胁迫。研究结果为进一步阐述S2P同源蛋白sll0862在集胞藻PCC6803中的功能奠定基础。     

浑球红细菌Rhodobacter sphaeroides吸氢酶（hup）基因簇的分离 …

以Ｒｈｏｄｏｂａｃｔｅｒ ｃａｐｓｕｌａｔｕｓ的ｈｕｐＳ‘Ｌ基因ＤＮＡ片段为探讨,通过Ｓｏｕｔｈｅｒｎ杂交,从构建的Ｒｈｏｄｏｂａｃｔｅｒ ｓｐｈａｅｒｏｉｄｅｓ ６０１基因库中调取ｈｕｐ基因。阳性克隆Ｃｏｓｍｉｄ１可与Ｈｕｐ突变株ＪＰ９１,ＲＣＣ８以及ＢＳＥ８互补,而Ｃｏｓｍｉｄ３和Ｃｏｓｍｉｄ９只能与ＢＳＥ８互补。试验所产生的接合转移子均恢复了吸氢酶的活性和自养生长能力。     

聚球藻7942高效泌氨突变种的获得及其泌氨、谷氨酰胺合成酶活性、光合和生长

将含有Ａｎａｂａｅｎａｓｐ．ＰＣＣ７１２０反义ｇｌｎＡ基因片段的穿梭表达质粒ｐＤＣ－ＡＴＧＳ转化单细胞蓝藻聚球藻Ｓｙｎｅ－ｃｈｏｃｏｃｃｕｓ　ｓｐ．ＰＣＣ７９４２,通过同源重组,外源ＤＮＡ定位整合到染色体上。经过抗菌素筛选,获得一种高效泌氨的Ｓｙｎｅｃhｏｃｏｃｃｕｓ　ｓｐ．７９４２突变种。将此突变种固定化在聚氨脂泡沫中后,定量测定其谷氨酰胺合成酶（ＧＳ）活性。结果表明,固定化后的突变藻培养９ｄ后泌氨活性比自由生活的野生藻高１５６倍,ＧＳ活性降低７３．６％;其生长速度与同条件下野生藻相近,７７Ｋ荧光光谱表明突变种固定化后光系统Ⅱ活性提高４４％。     

螺旋藻（Spirulina platensis）藻胆体在解离过程中荧光发射光谱和光能传递的研究

对螺旋藻（Ｓｐｉｒｕｌｉｎａｐｌａｔｅｎｓｉｓ）藻胆体在室温和７７Ｋ处于不同浓度磷缓冲溶液和不同解离时间的荧光发射光谱进行了研究。藻胆体在０．９ｍｏｌ／Ｌ磷酸缓冲溶液中,由于没有发生解离,光能传递效率高,在７７Ｋ荧光发射光谱中只有一个峰,位于６８７ｎｍ,属于别藻蓝蛋白－Ｂ。当藻胆体悬浮在０．３ｍｏｌ／Ｌ磷酸缓冲溶液中１分钟,７７Ｋ荧光光谱的主峰出现在６８４ｎｍ．又出现６５５ｎｍ和６６６ｎｍ荧光峰,它们依次属子Ｃ－藻蓝蛋白和别藻蓝蛋白。在２小时;６５５ｎｍ荧先峰成为主峰,６８４ｎｍ荧光峰为次峰,６６６ｎｍ荧光肩消失。这表明Ｃ－藻蓝蛋白所捕获的先能已不能传递给别藻蓝蛋白,但能传给别藻蓝蛋白－Ｂ。我们提出在螺旋藻藻胆体中存在两类Ｃ－藻蓝蛋白,一是与别藻蓝蛋白相连接,另一是与别藻蓝蛋白－Ｂ相连接。     

螺旋藻（Spirulina platensis）藻胆体在解离过程中荧光发射光谱和光能传递的研究

对螺旋藻（Ｓｐｉｒｕｌｉｎａｐｌａｔｅｎｓｉｓ）藻胆体在室温和７７Ｋ处于不同浓度磷缓冲溶液和不同解离时间的荧光发射光谱进行了研究。藻胆体在０．９ｍｏｌ／Ｌ磷酸缓冲溶液中,由于没有发生解离,光能传递效率高,在７７Ｋ荧光发射光谱中只有一个峰,位于６８７ｎｍ,属于别藻蓝蛋白－Ｂ。当藻胆体悬浮在０．３ｍｏｌ／Ｌ磷酸缓冲溶液中１分钟,７７Ｋ荧光光谱的主峰出现在６８４ｎｍ．又出现６５５ｎｍ和６６６ｎｍ荧光峰,它们依次属子Ｃ－藻蓝蛋白和别藻蓝蛋白。在２小时;６５５ｎｍ荧先峰成为主峰,６８４ｎｍ荧光峰为次峰,６６６ｎｍ荧光肩消失。这表明Ｃ－藻蓝蛋白所捕获的先能已不能传递给别藻蓝蛋白,但能传给别藻蓝蛋白－Ｂ。我们提出在螺旋藻藻胆体中存在两类Ｃ－藻蓝蛋白,一是与别藻蓝蛋白相连接,另一是与别藻蓝蛋白－Ｂ相连接。     

Cu^2＋对蓝藻Spirulina maxima光合作用的抑制机理

Ｃｕ＾２＋Ｓｍａｘｉｍａ的生长和光合放氧均有抑制作用;高浓度Ｃｕ＾２＋使藻细胞中藻胆体产生的特征光吸收和荥光显著下降,表明藻胆体是Ｃｕ＾２＋作用位点之一。Ｃｕ＾２＋可促使离体的藻蓝蛋白变性,使其光吸收和荧光减弱、荧光偏振度减小,而别藻蓝蛋白对Ｃｕ＾２＋可能通过对藻胆体中的藻蓝蛋白的作用,抑制藻胆体的光收传递。     

嗜热蓝藻优雅粘囊藻藻胆体的特性和别藻蓝蛋白的亚基组成

研究了优雅粘囊藻藻胆体（ＰＢＳ）的特性。从聚丙烯酰胺凝胶电泳（ＰＡＧＥ）、吸收光谱和二阶导数图谱可证明,它的ＰＢＳ含有一种红色藻胆蛋白,两种Ｃ－藻蓝蛋白和三种别藻蓝蛋白。但是,用ＰＡＧＥ和羟基磷灰石柱层析分离和纯化所得藻胆蛋白,一般仅得到一种Ｃ－ＰＣ和一种亚基组成特殊的别藻蓝蛋白ＡＰＣ。这种ＡＰＣ吸收光谱和荧光发射相同于已报告的ＡＦＣ６６０ｎｍ。但是它的亚基组成是（αα′ββ′）２而不是（α′α２β２β′）Ｌｃ１０或（αβ）３。     

Studies on Cyanidium caldarium Phycobiliprotein Pigment Mutants

Phycobiliprotein biosynthesis was investigated in four strains of the unicellular rhodophyte, Cyandium caldarium, with different pigment phenotypes. All strains were incapable of synthesizing phycobiliproteins when grown in the dark. Western blotting experiments showed that dark-grown cells of the wild-type and mutant GGB synthesized the α and β subunit polypeptides of allophyocyanin and phycocyanin after exposure to light for 24 hours, whereas cells of mutant IIIC and GGBY did not. Similarly, light promoted the appearance of allophycocyanin and phycocyanin mRNAs in the wild-type and GGB but not in IIIC and GGBY. However, Southern blots of restricted genomic DNA from the wild type, IIIC, GGBY, and GGB, all hybridized with heterologous phycobiliprotein gene probes and revealed that all four strains contained identical Pst, EcoRI, and Dral restriction fragments containing allophycocyanin and phycocyanin genes. Cells of the wild type and GGB incubated in the dark with the heme precursor. δ-aminolevulinate, synthesized allophycocyanin and phycocyanin apoproteins providing strong evidence for the role of a tetrapyrrole in regulation of phycobiliprotein gene expression. However, cells of IIIC and GGBY incubated in the dark with δ-aminolevulinate did not contain detectable quantities of allophycocyanin or phycocyanin apoproteins. The possible role of a tetrapyrrole in phycobiliprotein gene expression and basis for the genetic lesion in mutants IIIC and GGBY is discussed.     

Transposon insertion in genes coding for the biosynthesis of structural components of the Anabaena sp. phycobilisome

Anabaena sp. PCC 7120 mutants defective in phycobiliprotein biosynthesis or phycobilisome assembly were generated by transposon mutagenesis. Four mutants with grossly reduced content of the major phycobiliprotein, phycocyanin, were found to have insertions within the cpcBACDEFG1G2G3G4 operon coding for phycocyanin biosynthesis and assembly. The insertion in mutant B646 separated the promoter from the open reading frames and eliminated production of the phycocyanin (CpcA) and (CpcB) subunits. Insertion in cpcC in mutant B642 eliminated production of the L³⁶ _Rlinker polypeptide required for assembly of phycocyanin into the distal discs of the phycobilisome rod substructures. Mutants B64328 and B64407 had insertions, respectively, in cpcE and cpcF, genes coding for the subunits of the heterodimeric lyase which catalyzes the attachment of phycocyanobilin to the phycocyanin apo- subunit. Mutant SB12, often unable to survive under low light, was found to have an insertion in the apcE gene coding for the large core-membrane linker (L¹²⁸ _CM) that provides the scaffold for assembly of the phycobilisome core. DNA sequencing 3 of apcE revealed genes apcABC, coding for the and subunits of allophycocyanin and for the small core linker L^7.8 _C. Amino acid sequence comparisons showed that the ApcA and ApcB proteins are 37% identical and that each of these polypeptides is highly similar to corresponding polypeptides from the distantly related filamentous strains Calothrix sp. PCC7601 and Mastigocladus laminosus.     

CHARACTERIZATION OF PHYCOCYANIN-DEFICIENT PHYCOBILISOMES FROM A PIGMENT MUTANT OF PORPHYRIDIUM SP. (RHODOPHYTA)

The structure and function of phycobilisomes in the rhodophyte Porphyridium sp. were investigated by comparing the properties of the wild type with a pigment mutant called C12. When grown under low light, cells of C12 were bright orange, while wild-type cells were deep red. The results obtained from a characterization of purified phycobilisomes of the mutant C12 led us to propose the existence in Porphyridium sp. phycobilisomes of two types of rods, some containing only phycoerythrin and others containing phycoerythrin bound to phycocyanin, which is in turn linked to the core by the linker L_RC. By studying the partitioning of phycobiliproteins between phycobilisomes and pools of free phycobiliproteins, we found that phycocyanin in the C12 mutant was only present in the pool of free proteins and that its specific linker, L_RC, was totally absent. Phycoerythrin was present in the free pool and in the purified phycobilisomes as well. One of the three specific phycoerythrin linkers γ was missing. In light of the fact that in the C12 mutant, the linker L_RC is absent and that there is no phycocyanin bound to the phycobilisomes, we propose that the rods in the mutant contain only phycoerythrin. These phycobilisomes are nevertheless functional and exhibit an efficient excitation transfer from phycoerythrin directly to allophycocyanin. Electron microscopy showed the purified phycobilisomes of C12 to be less dense than those of the wild type. This change was attributed to the disappearance of the rods containing the combination phycocyanin/phycoerythrin. Light still regulates phycobiliprotein synthesis in the mutant, as shown by the change in the color of the culture, which turned green-yellow when cells were shifted from low light to high light growth conditions. Light also regulates the structure of the phycobilisomes, which have fewer rods under high light growth conditions.     

The Calvin cycle in cyanobacteria is regulated by CP12 via the NAD(H)/NADP(H) ratio under light/dark conditions

In Synechococcus PCC7942 cells grown in the dark, the concentrations of NAD(H) and NADP(H) were 128+/-2.5 and 483+/-4.0 microm, respectively, while those in the cells under light conditions were 100+/-5.0 and 649+/-7.0 microm, respectively. Analysis of gel filtration indicated that the change of the ratio of NADP(H) to NAD(H) in cyanobacterial cells under light/dark conditions controls the reversible dissociation of the PRK/CP12/GAPDH complex (approximately 520 kDa) consisting of phosphoribulokinase (PRK), CP12, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). S. 7942 CP12 lacked the two Cys residues essential for formation of the N-terminal peptide loop in the CP12 of higher plants, but the N-terminal region of S. 7942 CP12 had the ability to be associated with PRK. The growth of mutant cells in which the CP12 gene was disrupted by a kanamycin resistance cartridge gene was almost the same as that of wild-type cells under continuous light conditions. However, under the light/dark cycle (12 h/12 h), the growth of CP12-disrupted mutant cells was significantly inhibited compared with that of wild-type cells. The mutant cells showed a decreased rate of O2 consumption and an increased level of ribulose 1,5-bisphosphate compared with wild-type cells in the dark. These data suggest that under light and dark conditions, the oligomerization of CP12 with PRK and GAPDH regulates the activities of both enzymes and thus the carbon flow from the Calvin cycle to the oxidative pentose phosphate cycle.     

Characterization and transcript analysis of the major phycobiliprotein subunit genes from Aglaothamnion neglectum (Rhodophyta)

The genes encoding the and subunits of allophycocyanin, phycocyanin and phycoerythrin from the red alga Aglaothamnion neglectum were isolated and characterized. While the operons containing the different phycobiliprotein genes are dispersed on the plastid genome, the genes encoding the and subunits for each phycobiliprotein are contiguous. The subunit gene is 5 for both the phycocyanin and phycoerythrin operons, while the subunit gene is 5 for the allophycocyanin operon. The amino acid sequences of A. neglectum phycobiliproteins, as deduced from the nucleotide sequences of the genes, are 65–85% identical to analogous proteins from other red algae and cyanobacteria. The conserved nature of the plastid-encoded red algal and cyanobacterial phycobiliprotein genes supports the proposed origin of red algal plastids from cyanobacterial endosymbionts.Many environmental factors effect phycobilisome biosynthesis. The effect of both nutrient availability and light quantity on the level of A. neglectum phycobiliprotein subunits and the mRNA species encoding those subunits is described.     

Identification of a gene required for cis-to-trans carotene isomerization in carotenogenesis of the cyanobacterium Synechocystis sp. PCC 6803.

A disruptive mutant of the sll0033 gene of the cyanobacterium Synechocystis sp. PCC 6803 produced primarily cis carotenes and small amounts of all-trans carotenes, but no xanthophylls, under dark conditions. Under light conditions, however, it produced normal carotenoids, that were the same as those produced by wild-type cells grown under both light and dark conditions. When the mutant cells cultured under dark conditions were irradiated, cis-isomers of carotenes were converted to all-trans lycopene. These findings demonstrate that this gene, designated crtH, is involved in the isomerization of cis-carotenes to all-trans forms in dark conditions, and that cis-carotenes were also converted to all-trans forms under light conditions by photoisomerization.