水拉恩氏菌JZ-GX1产嗜铁素特性及其对林木病原菌的拮抗作用

【背景】嗜铁素被认为是一种具有开发和利用价值的新型生物活性物质,已被逐渐应用到植物病原菌的防治中。【目的】明确根际促生菌水拉恩氏菌(Rahnella aquatilis) JZ-GX1产嗜铁素最佳发酵培养条件,进一步探讨嗜铁素在防治植物根部病害中的潜在作用。【方法】采用摇瓶发酵法,通过铬天青(Chrome azurol S,CAS)检测分析,对影响JZ-GX1菌株嗜铁素分泌的几种发酵因子进行研究,并通过菌丝生长抑制速率法测定嗜铁素对两种林木病原菌的拮抗效果。【结果】以KMB为基础培养基,初始pH 8.0,装液量25 mL/50 mL,按1%接种量接种,在28°C下培养36 h可获得该菌株较高产量的嗜铁素;鉴定其嗜铁素类型为羧酸盐和异羟肟酸型的复合型铁载体;在最适条件下测得其发酵原液对樟疫霉(Phytophthoracinnamomi)和立枯丝核菌(Rhizoctoniasolani)的抑制率均达到100%。【结论】水拉恩氏菌JZ-GX1对碱性土壤上林木根部病害的防治具有较好的潜力。     

两株聚球藻伴生细菌分离纯化与促生功能鉴定

【背景】蓝藻周围存在伴生细菌,伴生细菌与蓝藻具有复杂的作用关系。【目的】研究淡水聚球藻伴生细菌对聚球藻生长的影响。【方法】采用高通量测序分析聚球藻伴生细菌多样性;平板划线法纯化聚球藻伴生细菌,通过形态观察结合16S rRNA基因序列同源性比对,对其种属关系进行确定;通过聚球藻和不同浓度伴生细菌共培养测定其叶绿素a浓度,分析伴生细菌对聚球藻生长的影响;采用种子发芽试验验证伴生细菌促生功能。【结果】淡水聚球藻伴生细菌优势菌属为产卟啉杆菌属(Porphyrobacter)、根瘤菌属(Rhizobium)、水单胞菌属(Aquimonas)和中慢生根瘤菌属(Mesorhizobium),从聚球藻分离获得了两株伴生细菌JQ1和JQ2,基于16S rRNA基因序列鉴定其分别属于Rhizobium和Peribacillus,通过在聚球藻与不同浓度伴生细菌共培养及水稻发芽试验验证,证明伴生细菌JQ1和JQ2在菌藻比例分别为5:1和15:1时具有促生作用,都对增强秧苗素质和根系发育有一定影响但JQ2与JQ1相比能显著提高水稻种子的发芽率。【结论】淡水聚球藻伴生细菌JQ1和JQ2在适宜的浓度均可显著促进聚球...     

新型聚球藻噬藻体Yong-L2-223的分离及基因组分析

【目的】噬藻体(cyanophages)是特异性侵染蓝藻(cyanobacteria)的病毒，广泛分布于各类水体中，在调节蓝藻种群动态和密度、推动生物地球水生生态系统循环中起着重要作用。本研究的目的在于分离、鉴定噬藻体。【方法】本研究以海洋聚球藻(Synechococcus sp.) PCC 7002为指示宿主，从淡水水样中分离培养一株新型噬藻体Yong-L2-223，对其进行了宿主范围实验、全基因组测序、基因功能注释和系统进化分析。【结果】针对31株供试蓝藻的宿主范围实验，结果除指示藻PCC 7002 [属于聚球藻目(Synechococcales)]外，Yong-L2-223能够感染2株淡水蓝藻，分别是来源于滇池的绿色微囊藻(Microcystis viridis) FACHB-1342 [属于色球藻目(Chroococcales)]和水华束丝藻(Aphanizomenon flos-aquae)FACHB-1209[属于念珠藻目(Nostocales)]。既可在高盐条件下感染海洋蓝藻，又可在低盐条件下感染淡水蓝藻，Yong-L2-223具有广盐性。透射电镜观察表明，Yong-L2...     

附子白绢病菌拮抗菌的鉴定、发酵条件优化及抑菌效果

【背景】附子白绢病是由齐整小核菌(Sclerotiumrolfsii)引起的一种土传细菌性病害,该病原菌严重影响附子的生产。【目的】筛选出对附子白绢病具有生防效果的菌株,并将其用于病害防治。【方法】利用平板涂布法和划线法从黄粉虫蛹体分离菌株,平板对峙法筛选对齐整小核菌具有较强拮抗能力的菌株,通过形态学观察、生理生化试验、16S rRNA基因测序分析确定其分类地位;以单因素试验对发酵条件进行优化,并初步测定拮抗菌的菌悬液、挥发性气体和发酵液对齐整小核菌菌丝的抑制效果。【结果】筛选出的拮抗菌株将其编号为N2,初步鉴定为暹罗芽孢杆菌(Bacillus siamensis);菌株N2最佳培养基配方(g/L)：胰蛋白胨10.0,酵母浸粉5.0,蔗糖20.0;最佳培养条件：初始pH 6.0,温度36℃,转速240 r/min,装液量30 mL,接菌量0.05%;其菌悬液、挥发性气体和发酵液均能有效抑制白绢病菌菌丝的生长;胞外酶测定结果显示该菌株可产生蛋白酶和纤维素酶,不产嗜铁素。【结论】菌株N2对附子白绢病具有较好的抑制效果,在生物防治中具有较好的应用潜力和较高的研究价值。     

双层平板法检测嗜盐古菌铁载体

采用双层平板法应用于嗜盐古菌铁载体的原位检测。双层平板的上层为不添加铁离子的嗜盐古菌培养基, 嗜盐古菌可在其上生长, 在缺铁胁迫下可向外界分泌铁载体; 下层为含有CAS检测液用于铁载体检测的琼脂。当上层平板生长的嗜盐古菌分泌的铁载体透过培养基渗透到下层检测琼脂后, 即可在下层检测平板上产生明显的特征性的铁载体螯合晕圈, 表明双层平板法在嗜盐古菌的铁载体检测中确实可行, 且较原有的嗜盐古菌铁载体检测方法简便、直接。     

聚球藻7002在光生物反应器中的光自养培养

通过对聚球藻7002在光生物反应器中的培养,研究了光强在聚球藻7002培养液中的衰减规律,得到了培养过程光强随藻细胞浓度和光程距离变化的关系式,即I=I₀exp［-(-0.0239+0.0777OD₇₅₀)·L］。并对培养过程特性及培养温度、外加CO₂浓度和光照强度对藻细胞生长的影响进行了较为详细的研究,得到了反应器中较为适宜的聚球藻7002的培养条件,藻细胞培养密度达到3.4g/L(干重),体积产率达到0.57g/(L·d)的较高水平。     

双层平板法检测嗜盐古菌铁载体

采用双层平板法应用于嗜盐古菌铁载体的原位检测.双层平板的上层为不添加铁离子的嗜盐古菌培养基,嗜盐古菌可在其上生长,在缺铁胁迫下可向外界分泌铁载体;下层为含有CAS检测液用于铁载体检测的琼脂.当上层平板生长的嗜盐古菌分泌的铁载体透过培养基渗透到下层检测琼脂后,即可在下层检测平板上产生明显的特征性的铁载体螯合晕圈,表明双层平板法在嗜盐古菌的铁载体检测中确实可行,且较原有的嗜盐古菌铁载体检测方法简便、直接.     

固定化过程对聚球藻生理生化特性的影响

【背景】聚球藻(Synechococcus)是一类生长于海水中的单细胞蓝细菌,因生长迅速被用来净化污水。为了降低成本,生产上采用了固定化措施,但聚球藻固定化后,细胞内的生理生化变化尚未见报道。【目的】研究聚球藻固定化后生理生化及净化能力的变化,为促进聚球藻的应用提供科学依据。【方法】以海藻酸钠和氯化钙为主要原料固定聚球藻;利用显微观察法计算聚球藻的生长速率;利用溶氧仪检测聚球藻的净光合效率;利用荧光法检测一氧化氮(NO)含量;利用分光光度计法检测叶绿素含量、蛋白含量、硝酸还原酶(NR)活性、Rubisco羧化酶活性及水质指标。【结果】固定化过程使聚球藻的最大比生长速率降低24.30%。固定化后聚球藻的净光合速率降低范围为9.10%-29.10%,但固定化过程对叶绿素含量没有明显的影响。固定化使聚球藻Rubisco羧化酶活性、NO含量和NR活性分别降低25.70%、32.10%和40.00%,使聚球藻去除红鳍东方鲀养殖废水中总氮、总磷、氨氮和亚硝酸盐的能力分别降低30.00%、17.70%、20.20%和21.20%,但对去除硝酸盐及化学需氧量(Chemical oxygen demand,COD)的能力没有影响。【结论】固定化过程抑制了聚球藻NR的活性,使其NO产量减少,NO通过转录后修饰的方式降低了光合作用关键酶Rubisco的活性。Rubisco的活性降低使光合效率降低,导致固定化聚球藻的比生长速率和净化污水的能力降低。     

转小鼠金属硫蛋白-Ⅰ基因聚球藻7002的生长潜力分析

以野生聚球藻7002为对照, 从室温吸收光谱、光合放氧速率、生长动力学参数以及气升式光生物反应器中的生长特性阐述了转小鼠金属硫蛋白-Ⅰ基因聚球藻7002的生长优势和培养潜力。结果表明: 转MT聚球藻室温可见光光谱吸收峰比野生藻略高; 最大净光合速率和饱和光强比野生藻高, 呼吸速率和补偿光强比野生藻低; 转MT聚球藻摇瓶培养的最大细胞浓度为野生藻的1.74倍, 具有较高的细胞生长速率; 气升式光生物反应器有利于转MT基因聚球藻生长潜力的发挥。     

转小鼠金属硫蛋白-Ⅰ基因聚球藻7002的生长潜力分析

以野生聚球藻7002为对照,从室温吸收光谱、光合放氧速率、生长动力学参数以及气升式光生物反应器中的生长特性阐述了转小鼠金属硫蛋白-Ⅰ基因聚球藻7002的生长优势和培养潜力.结果表明:转MT聚球藻室温可见光光谱吸收峰比野生藻略高;最大净光合速率和饱和光强比野生藻高,呼吸速率和补偿光强比野生藻低;转MT聚球藻摇瓶培养的最大细胞浓度为野生藻的1.74倍,具有较高的细胞生长速率;气升式光生物反应器有利于转MT基因聚球藻生长潜力的发挥.     

PHYSIOLOGICAL PROFILES OF SYNECHOCOCCUS (CYANOPHYCEAE) IN IRON-LIMITING CONTINUOUS CULTURES1

We examined the physiology and biochemistry associated with the iron-limited continuous culture of the halotolerant cyanobacterium Synechococcus PCC 7002. Biomass production, photosynthetic pigment levels, photosynthetic efficiency, and the production of hydroxamate- and catechol-type siderophores are reported for cells grown over a range of available iron concentrations. The relationship between the yield of Synechococcus PCC 7002 in iron-limited chemostats and the concentration of available iron was not linear. Synechococcus PCC 7002 expressed an inducible physiological response that led to alterations either in the cellular iron quotient or, more likely, in levels of available iron due to induced iron-scavenging processes. During iron limitation these cyanobacteria produced components consistent with the activation of a high-affinity iron transport system; both hydroxamate- and catechol-type siderophores were detected. Iron-limited Synechococcus PCC 7002 also reduced CO₂ fixation rates from luxury levels to a rate that matched the cellular growth rate, presenting interesting implications for oceanic carbon flux models.     

细胞周期蛋白依赖性激酶抑制剂Roscovitine对3种蓝藻生长和活性的影响

为了明确蓝藻中丝氨酸/苏氨酸激酶的功能是否与调控细胞的生长分裂相关,以丝状鱼腥藻7120、单细胞集胞藻6803和聚球藻7002为对象,利用OD750光吸收测定和MTT方法研究了不同浓度丝氨酸苏氨酸激酶抑制剂roscovitine对其生长和脱氢酶活性的影响。结果表明:4 h roscovitine处理后对鱼腥藻7120和集胞藻6803生长量影响不大,对聚球藻7002的生长有促进作用。4 h roscovitine的处理对鱼腥藻7120有浓度依赖的显著抑制活性,对集胞藻6803的活性无影响,但是却促进聚球藻7002的活性。药物作用4 d后,7120的生长和活性均显著降低,并有浓度效应;6803的生长量较对照减少,但活性变化不明显;聚球藻7002的生长和活性均未受影响。显微观察结果显示,roscovitine对3种细胞形态没有影响,但药物作用4 d后的7120藻丝体较短。结果表明丝氨酸/苏氨酸抑制剂roscovitine影响丝状藻7120的生长和活性。     

Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002.

A physical restriction map of the genome of the cyanobacterium Synechococcus sp. strain PCC 7002 was assembled from AscI, NotI, SalI, and SfiI digests of intact genomic DNA separated on a contour-clamped homogeneous electric field pulsed-field gel electrophoresis system. An average genome size of 2.7 x 10(6) bp was calculated from 21 NotI, 37 SalI, or 27 SfiI fragments obtained by the digestions. The genomic map was assembled by using three different strategies: linking clone analysis, pulsed-field fragment hybridization, and individual clone hybridization to singly and doubly restriction-digested large DNA fragments. The relative positions of 21 genes or operons were determined, and these data suggest that the gene order is not highly conserved between Synechococcus sp. strain PCC 7002 and Anabaena sp. strain PCC 7120.     

ISOLATION OF ccmKLMN GENES FROM THE MARINE CYANOBACTERIUM, SYNECHOCOCCUS SP. PCC7002 (CYANOPHYCEAE), AND EVIDENCE THAT CcmM IS ESSENTIAL FOR CARBOXYSOME ASSEMBLY

A high CO₂ requiring mutant of the marine cyanobacterium Synechococcus PCC7002 was generated using a random gene-tagging procedure. This mutant demonstrated a reduced photosynthetic affinity for inorganic carbon (C_i) and accumulated high internal levels of C_i that could not be used for photosynthesis. Analysis of the mutant genomic DNA showed that the mutagenesis had disrupted a cluster of genes involved in the cyanobacterial CO₂ concentrating mechanism (CCM), the so-called ccm genes. These characteristics are consistent with a cyanobacterial mutant with defects in carboxysome assembly and/or functioning. Further genomic analyses indicated that the genes of the Synechococcus PCC7002 operon, ccmKLMN , are structurally similar to those of two closely related cyanobacteria, Synechococcus PCC7942 and Synechocystis PCC6803. The Synechococcus PCC7002 ccmM gene, which encodes a polypeptide with a predicted size of 70 kDa, was the direct target of the mutagenesis event. The CcmM protein has two distinct regions: an N-terminal region that shows similarity to an archaeon gamma carbonic anhydrase and a C-terminal region that contains repeated domains demonstrating sequence similarity to the small subunit of Rubisco. Physiological analysis of a ccmM -defined mutant showed that these cells were essentially identical to the original mutant; they required high CO₂ concentrations for growth, they had a low photosynthetic affinity for C_i, and they internalized C_i to high levels. Moreover, ultrastructural examination showed that both the original and the defined mutants lack carboxysomes. Thus, our results demonstrate that the ccmM gene of Synechococcus PCC7002 encodes a polypeptide that is essential for carboxysome assembly and therefore for proper functioning of the cyanobacterial CCM.     

Thermal protection of the oxygen-evolving machinery by PsbU, an extrinsic protein of photosystem II, in Synechococcus species PCC 7002.

The evolution of oxygen is the reaction that is the most susceptible to heat in photosynthesis. We showed previously that, in the cyanobacterium Synechococcus sp. PCC 7002, some protein factors located on the thylakoid membranes are involved in the stabilization of this reaction against heat-induced inactivation, and we identified cytochrome C550 as one such factor (Y. Nishiyama, H. Hayashi, T. Watanabe, N. Murata [1994] Plant Physiol 105: 1313-1319). In the present study we purified another protein that appears to be essential for the stabilization of the oxygen-evolving machinery. The purified protein had an apparent molecular mass of 13 kD, and the gene encoding the 13-kD protein was cloned from Synechococcus sp. PCC 7002 and sequenced. The deduced amino acid sequence revealed that the protein was homologous to PsbU, an extrinsic protein of the photosystem II complex, which has been found in thermophilic species of cyanobacteria. Western analysis showed that the level of PsbU in thylakoid membranes was constant, regardless of the growth temperature. Our studies indicate that PsbU, a constituent of the photosystem II complex, protects the oxygen-evolving machinery against heat-induced inactivation.     

人表皮生长因子（hEGF）基因在蓝藻中的表达

人表皮生长因子（hEGF)是由53个氨基酸组成的蛋白,在临床上内服与外敷可促进内外表皮细胞的生长。将人工合成的hEGF基因连接到质粒pRL-489上,位于启动子psb下游。验证连接成功后,用三亲接合转移方法将载体pRL-hEGF导入聚球藻Synechococcus sp.PCC7002和鱼腥藻Anabeana sp.PCC7120。由于pRL-hEGF没有能在单细胞蓝藻中自主复制的复制子,通过筛选,hEGF在聚球藻7002中是整合到蓝藻染色体上进行表达的。用PCR扩增的方法在两种转基因藻中均检测到hEGF基因的存在。放射免疫分析证明,hEGF基因在两种转基因藻中均得到了表达。而且,在聚球藻7002中是采用分泌形式将表达产物分泌到培养液中。     

Phycobiliprotein methylation. Effect of the gamma-N-methylasparagine residue on energy transfer in phycocyanin and the phycobilisome

The phycobiliproteins contain a conserved unique modified residue, gamma-N-methylasparagine at beta-72. This study examines the consequences of this methylation for the structure and function of phycocyanin and of phycobilisomes. An assay for the protein asparagine methylase activity was developed using [methyl-3H]S-adenosylmethionine and apophycocyanin purified from Escherichia coli containing the genes for the alpha and beta subunits of phycocyanin from Synechococcus sp. PCC 7002 as substrates. This assay permitted the partial purification, from Synechococcus sp. PCC 6301, of the activity that methylates phycocyanin and allophycocyanin completely at residue beta-72. Using the methylase assay, two independent nitrosoguanidine-induced mutants of Synechococcus sp. PCC 7942 were isolated that do not exhibit detectable phycobiliprotein methylase activity. These mutants, designated pcm 1 and pcm 2, produce phycocyanin and allophycocyanin unmethylated at beta-72. The phycobiliproteins in these mutants are assembled into phycobilisomes and can be methylated in vitro by the partially purified methylase from Synechococcus sp. PCC 6301. The mutants produce phycobiliproteins in amounts comparable to those of wild-type and the mutant and wild-type phycocyanins are equivalent with respect to thermal stability profiles. Monomeric phycocyanins purified from these strains show small spectral shifts that correlate with the level of methylation. Phycobilisomes from the mutant strains exhibit defects in energy transfer, both in vivo and in vitro, that are also correlated with deficiencies in methylation. Unmethylated or undermethylated phycobilisomes show greater emission from phycocyanin and allophycocyanin and lower fluorescence emission quantum yields than do fully methylated particles. The results support the conclusion that the site-specific methylation of phycobiliproteins contributes significantly to the efficiency of directional energy transfer in the phycobilisome.