Characterization of nitrogen-fixing moderate halophilic cyanobacteria isolated from saline soils of Songnen Plain in China

Twenty out of 200 isolates of cyanobacteria mainly from saline soils of Songnen Plain of China were successfully grown on BG11 N-free medium. The nitrogen-fixing activity was then demonstrated for the 20 isolates in modified BG 11 medium using the acetylene reduction assay. All of them possessed appreciable nitrogenase activity （acetylene reduction） under non-saline conditions; however, at 5% NaCl only 60% of the isolates exhibited a high rate of this activity and 25% were completely negative under these conditions. The cyanobacteria isolates grew well in BG11 medium; nevertheless, growth of the majority of isolates was reduced by about 25-85% in the same medium containing 5% NaCl. Cellulolytic activity was detected in 50% of the 20 strains, amylolytic in 45%, and pectinolytic in 10%o of the isolates. The cyanobacteria isolates showed also enzymatic activity under saline conditions （6%）. The preliminary identification indicated that seven isolates were Nostoc, two were Microcystis, four were Oscillatoria, six were Anabaena, and one isolate was Synechococcus.     

盐胁迫对红鳞蒲桃苗木生长和生理特性的影响

【目的】评价盐胁迫下红鳞蒲桃(Syzygizan hancei)苗木的生理耐受性。【方法】选择当年生红鳞蒲桃盆栽苗木为材料,研究6种NaCl盐分浓度(0%、0.2%、0.4%、0.6%、0.8%、1.0%)下红鳞蒲桃幼苗高生长、地径生长、叶片质膜透性、游离脯氨酸含量、可溶性糖含量、过氧化氢酶(CAT)活性、叶绿素含量的变化特征。【结果】随着NaCl浓度的增加,红鳞蒲桃苗木的高增长量逐渐降低,在浓度0.4%时下降显著;NaCl浓度对苗木的地径增长量影响不大;细胞质膜透性对盐胁迫具有较强的耐性,NaCl浓度在0.8%以下时,叶片的相对电导率差异不显著;游离脯氨酸和可溶性糖含量在NaCl浓度为0.4%时显著增加,其后维持在一定水平;CAT酶活性对盐胁迫较为敏感,在浓度0.2%时即显著增强;叶绿素含量则随着NaCl浓度的增加表现出先升后降的变化趋势。【结论】0.4%NaCl浓度是影响红鳞蒲桃苗木生理特性的关键。     

植物促生菌对燕麦初生苗盐分胁迫下的促生效应

为提高日益盐渍化耕地的作物抗盐性,从盐生植物根际土中分离得到4株含ACC脱氨酶的植物促生菌(PGPR),并在限菌条件下考察其对燕麦初生苗盐分胁迫下的促生效应.结果表明,随盐分的升高,促生作用增大.在10 g/L NaCl盐分胁迫下,4菌株对燕麦初生苗均表现出显著的促生效应,但以假单胞菌属S1最显著,其根长、根鲜重和根干重比未接菌对照分别增加137.2%、138.1%和96.2%.4株PGPR的ACC脱氨酶活性与植物生长参数(根长、根鲜重、根干重和下胚轴长)之间具有极显著的正相关性(相关系数>0.93).接种含ACC脱氨酶活性的PGPR可以作为一种环境友好、经济实用的盐渍化土壤改良措施.     

Cloning and Characterization of the fecC Gene Necessary for Optimal Growth under Iron-Deficiency Conditions in the Cyanobacterium Anabaena sp. PCC 7120

The fecC gene encoding a putative iron (Ⅲ) dicitrate transporte rwas cloned from nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, and inactivated. The mutant grows normally in medium with NO3^- , NH1^- or without combined nitrogen. But in iron-deficient medium, the mutant grows slowly. Photosynthetic properties were compared between the mutant and the wildtype strain, the content of photosynthetic pigments in the mutant is lower than that of the wild-type. The results of RT-PCR experiments show that the fecC gene is expressed under iron-deficient conditions, but is not expressed under iron-replete conditions. These results revealed that fecC gene product is required for optimal growth under iron-deficient conditions in Anabaena sp. PCC 7120.     

Genetic diversity of rhizobial populations recovered from three Lotus species cultivated in the infra-arid Tunisian soils

Eighty-three bacterial strains isolated from root nodules of Lotus creticus, L. pusillus, and L. arabicus grown in infra-arid Tunisian soils were characterized using a polyphasic approach including phenotypic analysis, rep-PCR and PCR-RFLP analyses of the 16S rRNA gene. Phenotypically, all isolates are fast growers the majority of which grow at a pH of between 5.5 and 9. Most of the tested isolates tolerate NaCl concentrations from 1.39% to 3.48%. By rep-PCR fingerprinting, the genomic similarity varied from 30% to 98%. All tested isolates were clustered into 32 rep-PCR clusters at the similarity level of 80%. The genomic divergence of strains revealed by rep/PCR analysis appeared to be very important since a molecular polymorphism delimiting symbionts for each species of Lotus was identified. With the high-resolution of rep-PCR profiles of the isolates obtained using Pearson’s/UPGMA analysis, the isolates were resolved into 60 different profiling groups to undergo 16S ARDRA analyses. The analysis of all restriction fragments from each strain based on the UPGMA algorithm from the combined patterns showed that Lotus isolates are very diverse and that they were affiliated to Sinorhizobium, Rhizobium, and Mesorhizobium genera.     

Diversity and high nitrogenase activity of endophytic diazotrophs isolated from Oryza rufipogon Griff.

Diversity and nitrogenase activity of endophytic diazotrophs colonized in the wild rice Oryza rufipogon Griff grown in Boluo, Huilai County in Guangdong Province and Lingshui County in Hainan Province were studied. Thirty-seven isolates obtained from Oryza rufipogon were identified as putative endophytic nitrogen-fixing bacteria by ARA （acetylene reduction assay） test and further confirmed by PCR amplification of nifH gene fragments. All obtained strains have ARA activity and the same sized nifH gene fragments. Above the similarity level of 80%, the obtained isolates were assigned as Group Ⅰ to Ⅷ by the clustering of IS-PCR fingerprints. The SDS-PAGE whole-cell protein patterns were similar to those of IS-PCR fingerprints. Components and contents of fatty acid methyl esters （FAMEs） were used to differentiate the representative strains （Ls13, Ls8, BL1, BL12, HL6, Ls4） from Group Ⅰ to Group Ⅵ. The six representative strains showed significant difference in contents and components of cellular fatty acid methyl ester. 16S rDNA sequencing analysis showed that strains of Group Ⅰ to Ⅶ were located in Enterobacteraceae （y-proteobacteria）. Strains of Group Ⅰ and Group Ⅱ were closely related to Klebsiella sp.; Strain Ls8 of Group Ⅱ was a little far away from the genus of Pantoea （homology level 96% with Pantoea agglomerans）, which may represent a new species or genus in Enterobacteraceae; Strains of Groups Ⅳ and Ⅴ belonged to different Enterobacter sp.; Strain Ls4 and Ls 9 representing Group Ⅵ were close to Citrobacter amalonaticus with 98% sequence similarity; Strain Ls15 of Group Ⅶ showed 98% sequence identity with Pantoea sp.; Strains of Group Ⅷ were assigned to the genus Ideonella （β-proteobacteria）. Based on the above results, endophytic diazotrophs isolated from O. rufipogon showed great diversity and some diazotrophs showed high nitrogenase activity with 42.52 μmol/mL. h C2H4. Inoculation to rice tests indicated that the isolated endophytic diazotrophs significantly promoted the rice growth.     

Heterologous expression of amylase gene from Saccharomycopsis fibuligera in an industrial strain of Saccharomyces cerevisiae

An α-amylase encoding gene was amplified by polymerase chain reaction fromSaccharomycopsis fibuligera and inserted into a shuttle vector YEp352, together with the yeast phosphoglycerate kinase 1 promoter and α-factor signal gene. The recombinant expression plasmid pLA8α was transformed into an industrial strain ofSaccharomyces cerevisiae Sc-11. The activity of the α-amylase produced by the transformant Sc-11-pLA8α was 6.3 U/mL and the starch utilization rate in YPS medium was 42%. The purified amylase was analyzed by SDS-PAGE, showing a molecular weight of 55×10³ protein band. Furthermore, the residual sugar, ethanol and some volatile compounds in the fermented worts under simulating brewing conditions were determined by chromatographic analyses. The fermentation characteristics of Sc-11-pLA8α were similar to that of Sc-11 and only minor changes in the concentration of flavor compounds could be observed. Foundation item: Supported by the National Tenth Five-Year Hi-Technique Project (2001BA708B05-04). Biography: LIU Zeng-ran (1964-), fenale, Ph. D., research, direction: food and biotechnology.     

耐碳青霉烯酶肺炎克雷伯菌的分离鉴定及生物学特性分析

以临床尿路感染患者尿液为研究对象,通过分离培养及16s rDNA进行肺炎克雷伯菌的分离鉴定;采用Kirby-Bauer纸片法进行药敏实验,并对分离株进行碳青霉烯酶表型筛选;通过PCR检测常见的碳青霉烯酶耐药基因、荚膜血清型和毒力基因分布情况;对分离的肺炎克雷伯菌株进行了生物被膜形成能力及其对小鼠致病力的分析。本研究从采取的86例尿液标本中分离检出11株耐碳青霉烯酶肺炎克雷伯菌,分离率为12.79%。耐碳青霉烯酶基因PCR检测结果显示,blaNDM、blaVIM、blaIMP和blaKPC基因在分离株中均呈现不同程度的分布。耐药性分析发现11株肺炎克雷伯菌对氨苄西林耐药率为90.91%,对头孢噻肟耐药率为63.64%,对链霉素、庆大霉素、氯霉素和诺氟沙星的耐药率较低。耐碳青霉烯类肺炎克雷伯菌荚膜分型结果显示,分离的11株细菌中10株均为强毒力型菌株（90.9%）,其中K57血清型4株（36.4%）,K1血清型1株（9.1%）,K2血清型1株（9.1%）,K5血清型1株（9.1%）,K20血清型3株（27.3%）,提示该批分离株具有较强的致病力。此外,毒力因子分布结果显示,其毒力因子rmpA（54.5%）、Aerobactin F（54.5%）在菌株中分布较为广泛。生物被膜形成能力检测及小鼠致病性试验结果显示,9株分离株均有较强的生物被膜形成能力,菌株致病力可能与荚膜血清型及生物被膜形成能力相关。综上所述,临床分离的致尿路感染病原肺炎克雷伯菌呈现多重耐药特征,强毒力菌株以K57荚膜型为主,K1、K2均有分布,提示对尿路感染病原应加强耐药监测,合理使用抗菌药物,有效防控多重耐药和强毒力菌株的感染与流行。     

Ovicidal activity of two fungal pathogens （Hyphomycetes）against Tetranychus cinnabarinus （Acarina：Tetranichidae）

The carmine spider mite, Tetranychus cinna-barinus, is an economically important pest that devastates varieties of crops worldwide and develops significant resistance to common chemical pesticides, most of which lack ovicidal activity. In the present study, two isolates of entomopathogenic fungi, Beuaveria bassiana SG8702 and Pae-cilomyces fumosoroseus Pfrl53, were bioassayed against T. cinnabarinus eggs at 25 ℃ under a photophase of 12 : 12 (L:D). Infected eggs on Vicia faba var. minor leaves failed to hatch due to distortion and shrinkage and had fungal outgrowths when maintained under moist conditions. Sprays of B. bassiana conidia to T. cinnabarinus eggs (on leaves) at the concentrations of 58, 298 and 1306 conidia/mm2 (3 replicates per concentration and 35-65 fresh mite eggs per replicate) resulted in corrected egg mortalities of 20.4±4.2%, 36.0±7.6% and 64.6±12.5% (F=43.14, P <0.01), respectively; sprays of P. fumosoroseus at 129, 402 and 2328 conidia/mm2 caused egg mortalities of 16.1±11.1%,     

Specific degradation of photosystem II D1 protein by a protease （Air3815） in heterocysts of the cyanobacterium Anabaena sp. PCC7120

Some filamentous cyanobacteria form heterocysts under conditions lacking combined nitrogen for nitrogen fixation.Photosystem II is removed from heterocyst during the process of cell differentiation.Here,we demonstrate that Alr3815 is a protease that is capable of degrading D1 protein of photosystem II.Strain-322,which lacks alr3815,is impaired in nitrogen fixation in air because some oxygen evolving activity is retained in its heterocysts.Our results also suggest that calcium may play a regulatory role in D1 degradation during heterocyst differentiation.     

可降解蓖麻饼粕的蛋白酶产生菌筛选及产酶条件优化

利用酪素平板透明圈法初筛和测蛋白酶活力复筛,从分离自蓖麻饼粕和实验室保存的菌株中筛选出1株高产蛋白酶菌株P3-2,对其产酶条件进行了研究。结果表明,最适碳源为麦芽糖,最适氮源为蓖麻饼粕,Na+对产酶具有促进作用,培养基初始p H值为7.5时出现产酶高峰,最适接种量为4%,培养到36 h和84 h时有两个产酶高峰。经正交试验优化,得到较佳产酶培养基组成:2.0%麦芽糖、1.0%蓖麻饼粕及1.0%Na Cl。     

CW2M3分泌新型淀粉酶的最适条件及酶的应用条件

从原筛选的产酶菌株CW₂出发， 经多次紫外诱变、 初筛和复筛后， 获得了变异菌株CW₂M₃， 其产酶水平为原菌株的332.7％， 且具有良好的遗传稳定性. 薄层层析证明，CW₂M₃分泌的新型淀粉酶能有效地催化淀粉降解产生异麦芽低聚糖， 产物主要包括异麦芽糖、 潘糖、 异麦芽三糖等. 用正交法结合薄层层析法确定了各因素对CW₂M₃产酶水平的影响， 结果表明， 培养温度是显著因素，CW₂M₃的最适产酶条件为： 用牛肉膏培养基（牛肉膏0.7%、 蛋白胨0.7%、 NaCl 0.5%、 pH 7.0）培养， 温度40 ℃， 时间12　h. 用该酶催化淀粉转化为异麦芽低聚糖时， 酶促反应时间和温度均是显著影响因素， 酶催化淀粉降解的最适条件为: 温度65 ℃， 酶促反应1.0　h， 体系pH为7.0.     

渗透压对细菌的影响

在等渗溶液中,微生物正常生长繁殖;在高渗溶液中,细胞失水收缩,而水分为微生物生理生化反应所必需,失水会抑制其生长繁殖.根据不同盐质量分数下细菌的繁殖情况,可以判断渗透压对细菌的影响.用含不同质量分数NaCl的牛肉膏蛋白胨培养基培养细菌来观察渗透压对细菌的影响,选用的菌种为金黄色葡萄球菌,大肠杆菌,枯草芽孢杆菌.大肠杆菌耐高渗透压的能力较差,在质量分数为3%以下的NaCl溶液中能正常生长,在5%的NaCl溶液中受到抑制;枯草芽孢杆菌和金黄色葡萄球菌有较强的耐盐能力,枯草芽孢杆菌在5%的NaCl溶液中能正常生长,在10%的NaCl溶液中受到抑制,而金黄色葡萄球菌在10%的NaCl溶液中仍能正常生长,在高于10%的NaCl溶液中生长受到抑制.     

Salt tolerance conferred by over-expression ofOsNHX1 gene in Poplar 84K

OsNHX1 gene (Na⁺/H⁺ antiporter gene ofOryza sativa L.) was introduced into Poplar 84K withAgrobacterium tumefaciens- mediated transformation. PCR, Southern and Northern blot analysis showed thatOsNHX1 gene was incorporated successfully into the genome of Poplar 84K and expressed in these transgenic plants. Salt tolerance test showed that three lines of transgenic plants grew normally in the presence of 200 mmol/L NaCl, while the Na⁺ content in the leaves of the transgenic plants grown at 200 mmol/L NaCl was significantly higher than that in plants grown at 0 mmol/L NaCl. The osmotic potential in the transgenic plants with high salinity treatment was lower than that of control plants. Our results demonstrate the potential use of these transgenic plants for agricultural use in saline soils.     

酿酒酵母呼吸突变株的高糖胁迫反应研究

【目的】研究野生型甘蔗糖蜜发酵高产乙醇菌株MF1002及其糖分利用能力显著提高的呼吸突变菌株MF15c在高糖胁迫下的生理特性变化。【方法】测定在高糖胁迫下菌株的生长速率、出芽率、乙醇产量和超氧化物歧化酶(SOD)活力、过氧化氢酶活力、过氧化物酶活力以及细胞质和线粒体的ATP酶活力。【结果】在葡萄糖浓度分别为25%、30%和40%的高糖培养基中,MF15c菌株生长和乙醇发酵受抑制的程度均明显低于MF1002。当葡萄糖浓度为30%和40%时,MF15c的最大菌体数目、最高出芽率和乙醇发酵浓度等均显著高于MF1002。当葡萄糖浓度为30%时,两菌株胞内的SOD活力、过氧化氢酶活力、过氧化物酶活力以及细胞质和线粒体的ATP酶活力均显著上升。其中,MF15c的胞内SOD活力、胞内过氧化物酶活力、细胞质ATP酶活力和线粒体ATP酶活力在高糖胁迫下的上升幅度显著高于MF1002。【结论】MF15c较MF1002具有更强的高糖耐受能力。SOD活力、过氧化氢酶活力、过氧化物酶活力以及细胞质和线粒体的ATP酶均参与了两菌株的高糖胁迫反应,胞内SOD活力、胞内过氧化物酶活力、细胞质ATP酶活力和线粒体ATP酶活力可能与MF15c菌株的高糖耐受能力有关,可作为进一步改造该菌株的指导指标。     

Screening and denitrification characteristics of a slight halophilic heterotrophic nitrobacteria

A slight halophilic heterotrophic nitrobacteria named gs1 was separated from the matured activated sludge. According to the morphological observation,physiological biochemical tests and sequence analysis of the 16S rDNA,strain gs1 was identified to be as Pseudomonas sp. Sodium acetate and ammonium chloride were used as carbon and nitrogen sources,respectively,to investi-gate the characteristics of the bacterium. When cultured for 24 h under aerobic conditions,with the removal rates of the NH4+-N and COD being 82.2% and 74.73%,respectively,strain gs1 will have a nitrification function of producing NO2--N. When cultured for 24 h under aerobic conditions in nitrite medium,the removal rate of the NO2--N became 100%,and when cultured for 24 h under aerobic conditions in nitrate medium,the removal rate of the NO3--N became 97%. The result shows that this strain functions for either nitrification or denitrification,i.e.,it can complete the full process of biological deoxidation.     

Assaying poly(ADP-ribose) polymerase activity in plants by polarographic method

A new method has been developed to assay poly(ADP-ribose) polymerase (PARP) activity in plant tissues through determining the content of nicotinamide (NIC) produced by enzymatic reaction by linear sweeping polarographic method. The detection limit of NIC was 0.03μmol/L, the calibration graph was linear up to 5 Mmol/L ( r = 0.999). The recoveries were approximately in the range of 92% to 98% and the relative standard deviations were less than 6.6% . Moreover, NAD+ and other interference existing in the mixture after enzymatic reaction had been removed by simple pretreatment, thus PARP assays were not interfered. A rapid, simple, sensitive and reliable nonisotopic method is reported to assay PARP activity in plant tissues . The results show that the KmNAD+ value of PARP in maize ( Zea mays L.) seedlings is 59 and the optimum pH for PARP activity is 8.5. Moreover, physiological conditions affect PARP activity in plant tissues, which has not been reported previously. When tobacco ( Nico-tiana tobacum) suspension cells were stressed by NaCI at low concentrations (100, 200 mmol/ L), the PARP activity increased significantly; when the cells were stressed at high concentrations (400, 1 000 mmol/L), it decreased to or even below the control level. PARP activity in etiolated maize seedlings was higher than that in light-grown seedlings.     

Effects of acetylene at low concentrations on nitrification, mineralization and microbial biomass nitrogen concentrations in forest soils

Temperate forest surface soils at the varying distances from main trunks (e.g., Pinus koraiensis and Quercus mongolica) were used to study the effects of acetylene (C₂H₂) at low concentrations on nitrification, mineralization and microbial biomass N concentrations of the soils, and to assess the contribution of heterotrophic nitrification to nitrous oxide (N₂O) emissions from soils. The use of acetylene at partial pressures within a range from 10 to 100 Pa C₂H₂ in headspace gas gave a significant decrease in N₂O emission at soil moisture of c. 45% water-filled porosity space, and the decrease was almost the same in each soil after exposure of C₂H₂ at low concentrations. Heterotrophic nitrification could account for 21%―48% of total N₂O emission from each soil; the contribution would increase with increasing distances from the Pinus koraiensis trunks rather than from the Quercus mongolica trunks. Under the experimental conditions, the use of C₂H₂ at low concentrations showed no significant influence on soil microbial biomass N, net N mineralization and microbial respiration. However, 100 Pa C₂H₂ in headspace gas could reduce carbon dioxide (CO₂) emissions from soils. According to the rapid consumption of 10 Pa C₂H₂ by forest soils and convenience for laboratory incubations, 50 Pa C₂H₂ in headspace gas can be used to study the origin of N₂O emissions from forest soils under aerobic conditions and the key associated driving mechanisms. The N₂O and CO₂ emissions from the soils at the same distances from the Quercus mongolica trunks were larger than those from the Pinus koraiensis trunks, and both emissions decreased as the distances from trunks increased. The stepwise regression analysis showed that 95% of the variability in soil CO₂ emissions could be accounted for by the concentrations of soil total C and water soluble organic C and soil pH, and that 72% of the variability in soil N₂O emissions could be accounted for by the concentrations of soil total N, exchangeable NH⁺₄-N and microbial biomass N and 25% of the variability in heterotrophic nitrification by the soil microbial biomass N concentration. The emissions of N₂O and CO₂ from forest soils after exposure of C₂H₂ at low concentrations were positively related to the net nitrification of the soils.     

温度、光照及藻细胞密度对3种水华蓝藻生长及竞争的影响

以铜绿微囊藻(Microcystis aeruginosa Kutzing)、细小平裂藻(Merismopedia minima G.Beck)和土生伪鱼腥藻(Pseudanabaena mucicola(NaumannHuber-Pestalozzi)Schwabe 1964)为研究对象,运用单因子和正交实验的方法,分析了温度、光照以及藻细胞初始密度对三者生长及竞争的影响.结果表明:铜绿微囊藻和细小平裂藻均能在高温、高光照的条件下达到最佳生长状态;黏伪鱼腥藻只有在低温、低光照的情况下才具有竞争优势.在相同的温度和光照条件下,不同的藻细胞初始密度是其竞争获得优势藻种地位的关键因子.除了控制营养盐和其他污染物的输入外,关注浮游植物群落结构变化,控制和降低蓝藻在水华爆发前的生物量密度,是治理淡水湖泊(水库)蓝藻水华的有效方法.     

金花小檗功效成分及其抑菌和抗氧化活性的研究

为充分利用川西南民族药——金花小檗,用不同的方法对其抑菌及抗氧化活性进行了测定.用酸性染料比色法、 HPLC法测定了金花小檗根、茎、醇提物、萃取部位的功效组分含量,发现生物碱集中于正丁醇部位和水部位中,小檗碱在醇提物、萃取部位中的含量都最多,巴马汀最少.测定其根、茎、醇提物对金黄色葡萄球菌、大肠杆菌的抑菌作用可知:与75%乙醇相比,金花小檗根、茎、醇提物对金黄色葡萄球菌的抑菌效果为低敏;而与75%乙醇相比,金花小檗根、茎、醇提物对大肠杆菌的抑菌效果为中敏.用DPPH·清除率法、总还原力法测定其醇提物和萃取部位的抗氧化活性,结果相似:正丁醇部位、水相部位的抗氧化活性较强,石油醚萃取部位的抗氧化活性最低.用金花小檗醇提物、正丁醇-水部位分别饲喂小鼠30 d测定小鼠血清MDA和T-SOD值,所得结果与正常组比较,试验组小鼠T-SOD值显著降低, MDA值显著升高,说明金花小檗有显著的抗氧化活性.因此,金花小檗提取物、萃取部位不仅有显著的抑菌活性,而且也有较高的体、内外抗氧化活性.