橙色红曲菌As3.4384 orf7基因缺失株的构建及其功能分析

红曲菌能产生多种有益的次级代谢产物,但红曲菌也产生一种对人和哺乳动物肝和肾有毒害的毒素,即桔霉素。因此控制毒素的产生是保障红曲产品安全性所必须的。故对桔霉素的合成途径及相关的基因做深入了解。6个桔霉素合成相关的基因成簇位于21 kb的DNA片段上。克隆了一个新基因(orf7基因),其位于该基因簇的外侧。采用基因敲除技术,构建红曲菌orf7基因缺失菌株。并采用紫外分光光度法检测orf7基因缺失菌株的红曲色素产量,HPLC法检测其桔霉素产量。orf7缺失菌株产红曲色素能力与出发菌株As3.4384相比没有变化;产桔霉素培养13~19 d,桔霉素的产量与出发菌株As3.4384相比增加了 142.4%。从而证实orf7基因与桔霉素代谢相关。     

Effects of penicillin G on morphology and certain physiological parameters of Lactobacillus acidophilus ATCC 4356

Evidence shows that probiotic bacteria can undergo substantial structural and morphological changes in response to environmental stresses, including antibiotics. Therefore, this study investigated the effects of penicillin G (0.015, 0.03, and 0.06 mg/l) on the morphology and adhesion of Lactobacillus acidophilus ATCC 4356, including the colony morphotype, biofilm production, hydrophobicity, H?O? formation, S-layer structure, and slpA gene expression. Whereas only smooth colonies grew in the presence of penicillin, rough and smooth colony types were observed in the control group. L. acidophilus ATCC 4356 was found to be hydrophobic under normal conditions, yet its hydrophobicity decreased in the presence of the antibiotic. No biofilm was produced by the bacterium, despite testing a variety of different culture conditions; however, treatment with penicillin G (0.015-0.06 mg/l) significantly decreased its production of H?O? formation and altered the S-layer protein structure and slpA gene expression. The S-protein expression decreased with 0.015 mg/l penicillin G, yet increased with 0.03 and 0.06 mg/l penicillin G. In addition, the slpA gene expression decreased in the presence of 0.015 mg/l of the antibiotic. In conclusion, penicillin G was able to alter the S-layer protein production, slpA gene expression, and certain physicochemical properties of Lactobacillus acidophilus ATCC 4356.     

Production of citrinin by various species ofMonascus

Summary The production of citrinin by variousMonascus species was determinated using various culture mediums and conditions. The maximal production was obtained in fermentor usingM. ruber with concentrations of 380 mg/l. Since citrinin is a toxic product, it is essential that the production of red pigments as food additives fromMonascus sp. avoid the occurrence of citrinin; so, we argue that some nitrogen sources are unfavorable to the production of citrinin.     

Production of bioactive phenolic acids and furanocoumarins in in vitro cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams. under different light conditions

Extracts from the biomass of Ruta graveolens and Ruta graveolens ssp. divaricata cultured in vitro under different light conditions (far-red, red and blue light, UV-A irradiation, in darkness and white light) were tested for the amounts of free phenolic acids and cinnamic acid (twelve compounds) as well as furanocoumarins and umbelliferone (seven compounds) using HPLC methods. Total amounts of the investigated groups of compounds in the cultures of both plants increased from 2.6 to 6.7 times, depending on light quality, and the maximum values reached were 106.50 and 1,276.74?mg?100?g^?1 DW (in R. graveolens), and 106.97 and 262.54?mg?100?g^?1 DW (in the subspecies), respectively. Both white light and blue light were equally beneficial for the total production of phenolic acids in cultures of both plants, whereas the total production of furanocoumarins was clearly better stimulated by blue light in R. graveolens and by darkness in the subspecies (i.e. the amounts were respectively 1.44 and 1.7 times higher than in the biomass cultivated under white light). The amounts of individual compounds in both plant cultures increased from about 2.2 to 26.3 times depending on light quality. The following bioactive compounds were obtained in quantities which are of interest from a practical perspective: in R. graveolens culture??protocatechuic acid (45?mg?100?g^?1 DW), isopimpinellin (about 500?mg?100?g^?1 DW) and bergapten (about 270?mg?100?g^?1 DW), and in the subspecies culture: p-coumaric acid (70?mg?100?g^?1 DW) and isopimpinellin (about 210?mg?100?g^?1 DW).     

The differentially expressed C21orf5 gene in the medial temporal-lobe system could play a role in mental retardation in Down syndrome and transgenic mice

Mental retardation represents the more invalidating pathological aspect of Down syndrome, DS, and has a hard impact in public health. Modifications in DS brain, concerning abnormal size, neuronal differentiation, and cell density, cause changes in the neurophysiology and behavior of DS patients, and could be determined by dosage imbalance of genes localized in the DS critical region, DCR. Among these genes, C21orf5 showed high homology with Caenorhabditis elegans Pad1 involved in cellular differentiation and patterning. To shed light on C21orf5 role in DS, we performed molecular characterization of human and mouse orthologs, their spatio-temporal expression during development and in adult, and overexpression in DS and transgenic mice. C21orf5 was widely expressed early in embryogenesis in the nervous system. Later, its expression became differential and increased in mesencephalon and rhomboencephalon. This developmental expression profile evolves selectively in adult brain with higher signals in hippocampus, cerebellum, perirhinal, and entorhinal cortex, compared to the other cortical regions. Cellular specificity was detected in hippocampus with higher C21orf5 mRNA level in CA3 cells. Our findings appoint C21orf5 as candidate gene for mental retardation: Its overexpression in DS cells may contribute to gene imbalance in DS.Its specific expression in normal and its mirroring pattern in transgenic mice correspond to abnormal regions in DS patients and to neurological phenotype of transgenic mice. Altered cortical lamination in transgenic mice and the Pad1 ortholog function suggest a potential role of C21orf5 in cell differentiation. Its patterned differential expression in the medial temporal-lobe system, including hippocampal formation and perirhinal cortex involved in memory storage, and learning and memory defects in the transgenic mice suggest a specialized role for C21orf5 in cognitive processes. These evidences suggest that C21orf5 is an attractive candidate gene contributing to neurological alterations responsible for mental retardation in DS patients.     

Improvement of monacolin K, γ-aminobutyric acid and citrinin production ratio as a function of environmental conditions of<Emphasis Type="Italic"> Monascus purpureus</Emphasis> NTU 601

Monascus, a traditional Chinese fermentation fungus, is used as a natural dietary supplement. Its metabolic products monacolin K and -aminobutyric acid (GABA) have each been proven to be a cholesterol-lowering drug and a hypotensive agent. Citrinin, another secondary metabolite, is toxic to humans, thus lowering the acceptability of red mold rice to the general public. In this study, the influence of different carbon and nitrogen sources, and fatty acid or oils, on the production of monacolin K, citrinin and GABA by Monascus purpureus NTU 601 was studied. When 0.5% ethanol was added to the culture medium, the production of citrinin decreased from 813 ppb to 561 ppb while monacolin K increased from 136 mg/kg to 383 mg/kg and GABA increased from 1,060 mg/kg to 7,453 mg/kg. In addition, response surface methodology was used to optimize culture conditions for monacolin K, citrinin and GABA production, and data were collected according to a three-factor (temperature, ethanol concentration and amount of water supplemented), three-level central composite design. When 500 g rice was used as a solid substrate with 120 ml water and 0.3% ethanol, the production of monacolin K at 30°C increased from 136 mg/kg to 530 mg/kg, GABA production increased from 1,060 mg/kg to 5,004 mg/kg and citrinin decreased from 813 ppb to 460 ppb.     

不同波长光源对新蚜虫疠霉产孢能力的影响及霉菌蓝光受体基因的克隆和表达分析

光在调控真菌的多种生理过程中发挥着重要作用。为探究光照对新蚜虫疠霉Pandora neoaphidis产孢的影响,本文研究了不同波长光源(蓝光、绿光、白光、红光、黄光)和无光黑暗条件对新蚜虫疠霉分生孢子弹射能力的影响,通过cDNA末端的快速扩增(RACE)对新蚜虫疠霉的蓝光受体蛋白基因pnwc-1进行克隆并对其进行生物信息学分析,利用qRT-PCR对蓝光光源不同照射时长下pnwc-1的表达量进行了定量分析。结果表明,蓝色光源(波长460–465nm)照射后的新蚜虫疠霉菌丝产生的分生孢子数量显著高于其他波长光源,排序为:蓝光>绿光>白光>红光>黄光>无光。另外,分析克隆获得的全长为2 423bp的pnwc-1基因发现,其编码的蛋白具有蓝光受体蛋白典型的保守结构域,同源比对结果显示新蚜虫疠霉与接合菌门真菌归为一类但相对独立。qRT-PCR的定量分析结果表明随着照射时长增加,蓝光处理能显著提高pnwc-1的表达量,而且pnwc-1的相对表达量与累积产孢量存在正相关(R2=0.9798)。本研究为后续蓝光及其受体基因功能的深入研究提供了实验基础,并促进以新蚜虫疠霉为代表的虫霉目真菌在害虫生物防治中的应用。     

Effects of phosphorus concentration and light intensity on the biomass composition of Arthrospira (Spirulina) platensis

This paper presents the effects of various phosphorus concentrations (10, 50, 250 and 500 mg l(-1) K(2)HPO(4)) on the biomass production and composition of Arthrospira (Spirulina) platensis in relation to light intensity (24, 42 and 60 μE m(-2) s(-1)). The maximum biomass production was 3,592 ± 392 mg l(-1) and this was observed in 250 mg l(-1) K(2)HPO(4) at 60 μE m(-2) s(-1) light intensity after 32 days of cultivation. A maximum specific growth rate (μ(max)) of 0.55 d(-1) was obtained in 500 mg l(-1) K(2)HPO(4) at 60 μE m(-2) s(-1). The protein, lipid and chlorophyll contents of the biomass varied from 33.59 to 60.57 %, 5.34 to 13.33 % and 0.78 to 2.00 %, respectively. The most significant finding was that phosphorus limitation (10 mg l(-1) K(2)HPO(4)) caused a drastic increase of the carbohydrate content (59.64 %). The effect of phosphorus limitation on the carbohydrate content was independent of the light intensity. The accumulated carbohydrates are proposed to be used as substrate for biofuel generation via one of the appropriate biomass energy conversion technologies. Also, it was observed that phosphorus removal is a function of biomass density, phosphorus concentration and light intensity.     

A novel negative regulatory factor for nematicidal Cry protein gene expression in Bacillus thuringiensis

A 3-kb HindIII fragment bearing the cry6Aa2 gene and the adjacent and intergenic regions was cloned from Bacillus thuringiensis strain YBT-1518. Two open reading frames (ORFs), namely, orf1 (termed cry6Aa2) and orf2 that were separated by an inverted-repeat sequence were identified. orf1 encoded a 54-kDa protein that exhibited high toxicity to the plant-parasitic nematode Meloidogyne hapla. The orf2 expression product was not detected by SDS-PAGE, but its mRNA was detected by RT-PCR. The orf2 coexpressed with orf1 at a high level in the absence of the inverted-repeat sequence, whereas, the expression level of orf1 was decreased. When orf2 was mutated, the level of orf1 expression was enhanced obviously. In conclusion, the inverted-repeat sequence disturbs orf2 expression, and the orf2 downregulates orf1 expression. This is an example of novel negative regulation in B. thuringiensis and a potential method for enhancing the expression level of cry genes.     

Characterization of the urease gene cluster from Rhizobium leguminosarum bv. viciae

Moderate levels of urease activity (ca. 300 mU mg(-1)) were detected in Rhizobium leguminosarum bv. viciae UPM791 vegetative cells. This activity did not require urea for induction and was partially repressed by the addition of ammonium into the medium. Lower levels of urease activity (ca. 100 mU mg(-1)) were detected also in pea bacteroids. A DNA region of ca. 9 kb containing the urease structural genes ( ureA, ureB and ureC), accessory genes ( ureD, ureE, ureF, and ureG), and five additional ORFs ( orf83, orf135, orf207, orf223, and orf287) encoding proteins of unknown function was sequenced. Three of these ORFs ( orf83, orf135 and orf207) have a homologous counterpart in a gene cluster from Sinorhizobium meliloti, reported to be involved in urease and hydrogenase activities. R. leguminosarum mutant strains carrying Tn 5 insertions within this region exhibited a urease-negative phenotype, but induced wild-type levels of hydrogenase and nitrogenase activities in bacteroids. orf287 encodes a potential transmembrane protein with a C-terminal GGDEF domain. A mutant affected in orf287 exhibited normal levels of urease activity in culture cells. Experiments aimed at cross-complementing Ni-binding proteins required for urease and hydrogenase synthesis (UreE and HypB, respectively) indicated that these two proteins are not functionally interchangeable in R. leguminosarum.