蛋白和酯酶电泳在根霉鉴定上的应用

对根霉(Rhizopus)属的十个种或变种共二十四株菌的菌体可溶性蛋白和酯酶同工酶进行了电泳的研究得到对这个属分类上更多的依据.在严格控制培养,提取和电泳条件的情况下,同一株菌不同批次所得菌体蛋白电泳图谱有较好的重复性.在相同的条件下,每个种的根霉有各自特征性蛋白图谱,种内不同菌株的蛋白图谱和酯酶酶谱基本相同.特别是形态特征明显、分类地位明确的种,种内各株的图谱也较一致,如R. stolonifer;与R.circinans.在确定新变种R. delemar var. latoapicalis时,电泳图谱与R.delemar var.delema:有明显不同,起到了佐证作用.因此认为,蛋白图谱与酯酶酶谱相辅相成,在根霉种的分类中是一有效的辅助手段.     

Purification and characterization of a novel protease-resistant α-galactosidase from Rhizopus sp. F78 ACCC 30795

A novel extracellular α-galactosidase, named Aga-F78, from Rhizopus sp. F78 ACCC 30795 was induced, purified and characterized in this study. This soybean-inducible α-galactosidase was purified to homogeneity by ammonium sulfate precipitation and fast protein liquid chromatography (FPLC), with a yield of 14.6% and a final specific activity of 74.6 U mg⁻¹. Aga-F78 has an estimated relative molecular mass of 78 kDa from SDS-PAGE while native mass of 210 kDa and 480 kDa from non-denaturing gradient PAGE. This α-galactosidase had no N- or O-glycosylated. Amino acid sequences of three internal fragments were determined, and fragment 1, NQLVLDLTR, shared high homology with bacterial and fungal GH-36 α-galactosidases. The optimum pH and temperature on activity of Aga-F78 were 4.8 and 50 °C, respectively. The properties of pH and temperature stability, effect of ions and chemicals were also studied. Furthermore, the resistant to neutral and alkaline proteases and substrate specificity of natural substrates (melibiose, raffinose, stachyose and guar gum) were also studied to enlarged the application of Aga-F78 in more fields. Kinetic studies revealed a K_m and V_max of 2.9 mmol l⁻¹ and 246.1 μmol (mg min)⁻¹, respectively, using pNPG as substrate. To our knowledge, this is the first report of purification and characterization of α-galactosidase from Rhizopus with some special properties, which may aid its utilization in the food and feed industries.     

Field and laboratory studies on the fungus Aspergillus parasiticus, a pathogen of the pink sugar cane mealybug Saccharicoccus sacchari

Infestation of sugar cane nodes by the mealybug Saccharicoccus sacchari (Cockerell) was studied in two commercial fields over a 7-month period in 1987. Natural enemies associated with S. sacchari were fungi Aspergillus parasiticus Speare, Metarhizium anisopliae (Metschnikoff) Sorokin, and Penicillium spp.; the dipteran Cacoxenus perspicax Knab; and the hymenopteran parasitoid Anagyrus saccharicola Timberlake. A. parasiticus was the predominent natural enemy of S. sacchari whereas all other natural enemies showed a low level of activity. The highest prevalence of A. parasiticus was in March when it occurred on 84% of S. sacchari-infested nodes. The prevalence of A. parasiticus declined rapidly during April and May and was absent in the winter months during which nodal infestation of S. sacchari increased. In laboratory bioassays all fungal isolates originating from S. sacchari were more virulent at 28°C than at 24°C. Laboratory studies supported the hypothesis based on field observations that temperature highly influenced the efficacy of A. parasiticus against S. sacchari.     

In vitro pathogenicity assay for the ergot fungus Claviceps purpurea

The pathogenic development of the biotrophic ergot fungus Claviceps purpurea is strictly limited to the ovary of grasses. Early colonization stages occur within a defined spatio-temporal course of events, including the directed growth to the vascular tissue for nutrient supply. To characterize mutant strains with putative defects in pathogenicity, the close observation of the infection pathway is therefore indispensable. Here, we describe the establishment of a new pathogenicity assay, based on the in vitro cultivation of isolated rye ovaries. The pathogenic development of a wild-type strain of C. purpurea was compared with the infection of mature rye flowers on whole plants. Up to the sixth day post inoculation, the route of infection within the isolated ovaries was maintained and temporally equal to that seen in mature flowers. Therefore, the in vitro pathogenicity assay is an effective alternative to the whole-plant infection tests, and suitable for detailed infection studies and screening high numbers of mutants for defects in early pathogenesis.     

The halotolerant fungus Glomerobolus gelineus is a member of the Ostropales

Glomerobolus gelineus is a halotolerant species with a unique method of ballistic propagation. The absence of both sexual and asexual spores made reliable placement of this species, based on morphology alone, within the current fungal classification problematical. A phylogenetic analysis of the large and small nuclear ribosomal subunit and the second largest subunit of RNA polymerase II placed this fungus within the Ostropales, an order comprising lichenized and saprobic species, with good statistical support. Subsequently, a more detailed analysis that combined the nuc LSU rDNA and the mt SSU rDNA confirmed a close relationship to the Stictidaceae. The phylogenetic placement of G. gelineus is also supported by morphological characters. We postulate that the hyphoma lobes of Glomerobolus correspond to the periphysoidal layer in the apothecium of Stictis, and the propagule to the hymenium. Moreover, the presence of crystals in the outer lobes of G. gelineus is another indication of its relationship with Ostropales, which have characteristic crystalliferous hyphae. The placement of Glomerobolus within the Ostropales further expands the ecological diversity exhibited by this order. It also provides a phylogenetic hypothesis for assessing the homology of the enigmatic hyphomal morphology with apothecia-forming Ascomycota.     

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10⁵ CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and β-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.     

玫烟色棒束孢(Isaria fumosorosea)侵染小菜蛾的表达谱分析

【背景】昆虫病原真菌对寄主的侵染是一个十分复杂的过程,是多基因共同作用的结果。玫烟色棒束孢(Isaria fumosorosea) IFCF01菌株对小菜蛾具有很高的致病力,然而有关玫烟色棒束孢对小菜蛾致病的相关基因少见报道。【目的】筛选玫烟色棒束孢侵染小菜蛾相关基因,为更好地利用玫烟色棒束孢防治小菜蛾提供基因靶点。【方法】采用第二代高通量测序技术RNA-Seq,对玫烟色棒束孢侵染小菜蛾2–3龄幼虫4、8、12、16、24、30、36 h的虫菌混合样品(处理组)及纯培养玫烟色棒束孢(对照组)进行测序分析并筛选差异表达基因,结合生物信息学方法分析差异基因涉及的功能模块和信号通路。【结果】玫烟色棒束孢侵染小菜蛾混合样品与纯培养玫烟色棒束孢对照组对比分析共获得28 384个差异基因,其中显著差异表达基因274个,上调表达118个,下调表达156个。筛选获得的显著差异表达基因,特别是上调表达基因可能与玫烟色棒束孢对小菜蛾的侵染有关。GO二级分类显示,差异表达基因能够注释到36个GO条目中,包含18个生物学过程、9个细胞组分和9个分子功能。KEGG通路分析显示共有171个差异表达基因(differentially expressed gene,DEG)注释到132个通路中,其中有66个DEG显著富集在14个通路中。这些显著差异表达基因中大部分为玫烟色棒束孢侵染过程中潜在致病毒力相关基因。【结论】本研究为筛选玫烟色棒束孢侵染小菜蛾致病相关基因提供重要数据库,也为阐明玫烟色棒束孢对小菜蛾的侵染机制提供基础。     

Isariotins G–J from cultures of the Lepidoptera pathogenic fungus Isaria tenuipes

Four new alkaloids isariotins G–J (1–4), together with the known isariotins, were isolated from cultures of the Lepidoptera pathogenic fungus Isaria tenuipes. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. Compounds 1–4 exhibited antimalarial and cytotoxic activities.     

The Mycobacterium tuberculosis mysB gene product is a functional equivalent of the Escherichia coli sigma factor, KatF

Mycobacterium tuberculosis, the causative agent of tuberculosis, may remain dormant within its host for many years. The nature of this dormant or latent state is not known, but it may be a specialized form of the stationary growth phase. In Escherichia coli, KatF (or RpoS) is the major stationary phase sigma factor regulating an array of genes expressed in this phase of growth. A potential M. tuberculosis katF homologue was cloned using a fragment of the E. coli katF gene as a probe. DNA sequence analysis of a resultant clone showed 100% identity to a fragment of DNA encoding the M. tuberculosis mysA and mysB genes. Overexpression of mysB in M. bovis BCG resulted in an increase in katG mRNA and catalase and peroxidase activity, and an increase in sensitivity of the cells to isoniazid. An increase in katG promoter activity from a reporter vector was demonstrated when mysB was overexpressed from the same plasmid, indicating a direct relationship between MysB and katG expression.     

Potential of Lecanicillium species for dual microbial control of aphids and the cucumber powdery mildew fungus, Sphaerotheca fuliginea

Detached leaf disc bioassays were conducted against cucumber powdery mildew and three species of aphid with three entomopathogenic species of Lecanicillium; Lecanicillium longisporum (Vertalec^®), Lecanicillium attenuatum (CS625), and an unidentified isolate (DAOM198499). The three Lecanicillium species had high virulence against the aphids Myzus persicae, Macrosiphum euphorbiae and Aulacorthum solani with the exception of DAOM 198499, which demonstrated reduced virulence to A. solani with an LT₅₀ of 6.4 days. Otherwise, LT₅₀ ranged between two and four days. Suspensions of conidia and blastospores of the Lecanicillium species were also applied onto 15 mm leaf discs dissected from cucumber plants previously inoculated with Sphaerotheca fuliginea. Powdery mildew did not develop when the Lecanicillium applications were made one and eight days after S. fuliginea inoculations. When Lecanicillium was applied to highly infected leaf discs 11 and 15 days after S. fuliginea inoculation, the application suppressed subsequent production of S. fuliginea spores as compared to the controls. These results suggest the potential of a dual role for Lecanicillium spp. as microbial control agents against aphids and powdery mildew.     

The effects of temperature and chorionic gonadotropin on the functional properties of lactate dehydrogenase from oocytes of loach Misgurnus fossilis

Acclimation of loach to relatively low and high temperatures gives rise to changes in the properties of LDH from oocytes. The minimum of apparent K_m values for pyruvate of LDH from immature oocytes of fish acclimated to low temperature was registered at low assay temperatures, whereas adaptation to high temperatures leads to the enzyme showing a minimum at high temperatures. In thermal acclimation of fish the K_m minimum value drifts during 15 days. During oocyte maturation, the K_m minimum drifts during 40 hr. The differences in the kinetic features of LDH from oocytes both at thermal acclimation and during oocyte maturation are not associated with the appearance of new isozymes.     

A biocatalyst for pyruvate preparation from -lactate: lactate oxidase in a Pseudomonas sp.

For the purpose of producing pyruvate from -lactate by enzymatic methods, four microorganism strains that produce lactate oxidase (LOD) were screened and isolated from many soil samples. Among them, strain SM-6, which showed high potential for pyruvate production, was chosen for further research. Physiological studies and 16S rDNA relationship reveal that SM-6 belongs to Pseudomonas putida. The optimized pH and temperature of the enzyme-catalyzed reaction were pH 7.2, and 39 °C, respectively. Low-concentration EDTA (1 mM) could improve the stability of pyruvate and conversion ratio of lactate oxidase. V_max and K_m value for -lactate were 2.46 μmol/(min mg) protein and 9.53 mM, respectively. On preparation scale, cell-free extract from SM-6, containing 300 mg/l of crude enzyme (4037 U/ml lactate oxidase), could convert 66% of 116 mM of -lactate into 76.6 mM pyruvate in 18 h, and 82% of substrate was transformed after 48 h, giving 95.0 mM (10.5 mg/ml) of pyruvate. The ratio of product to biocatalyst was 34.8:1 (g/g).     

An unusual fructose 1,6-bisphosphate-activated lactate dehydrogenase from the ascidian Pyura stolonifera

Lactate dehydrogenase (LDH) was purified from the siphon muscle of the intertidal ascidian Pyura stolonifera. The enzyme is unique among chordate LDHs but resembles some bacterial and platyhelminth LDHs in being activated by fructose 1,6-bisphosphate (FBP). Concentrations of FBP in the range 5μM to 0.5 mM increase V_max of the pyruvate reductase reaction by 130% to 210%, and decrease K_m pyruvate 5 to 11 fold and K_m NADH 2.5 to 5 fold. The enzyme is also activated by inorganic phosphate, but requires a 50 fold higher concentration to attain the maximum activation achieved by 0.5 mM FBP. Of a range of metabolites tested, including other glycolytic sugar phosphates, only FBP and inorganic phosphate activated the enzyme. FBP activation was not observed with 16 representative vertebrate LDH homotetramers, but did occur to a limited extent with LDH from an echinoderm. LDH was the only pyruvate reductase enzyme detected in P. stolonifera siphon muscle, and its activity was much greater than that of phosphorylase or phosphofructokinase. The LDH reaction is utilized by P. Stolonifera during prolonged siphon closure on exposure to air when lactate, but not succinate, accumulates in the siphon muscle. While the ascidian enzyme provides the first example of a FBP activated LDH from a chordate, it remains to be determined if this unusual property has any role in metabolic regulation.     

Analysis of the Complete Nucleotide Sequence of the Tetracycline-Resistance Transposon Tn10

An analysis of the complete nucleotide sequence of the composite tetracycline-resistance transposon Tn10 (9147 bp) from the Salmonella typhi conjugative plasmid R27 is presented. A comparison of the protein sequences from IS10-right and IS10-left transposases has identified four amino acid differences. These residues appear to play an important role in normal transposase function and may account for the differences in exhibited transposition activities. The tetracycline determinants encoded by this version of Tn10 share >99% identity with those of Tn10^R100, demonstrating the conservation that exists between these transposons. A previously uncharacterized 3000-bp region of Tn10 contains four putative open reading frames. One of these open reading frames shares 55% identity with the glutamate permease protein sequence from Haemophilus influenzae although it was unable to complement an Escherichia coli glutamate permease mutant, with which it shares 51% identity. The three remaining putative open reading frames are arranged as a discrete genetic unit adjacent to the glutamate permease homolog and are transcribed in the opposite direction. Two of these open reading frames are homologous with Bacillus subtilis proteins of unknown functions while the other has no homologs in the database. The presence of an aminoacyl-tRNA synthetase class II motif in one of these open reading frames in combination with the glutamate permease homolog allows us to postulate that this region of Tn10 could once have played a role in amino acid metabolism.     

马尾松PmPGK1和PmGPIC基因的克隆和表达分析

为了解马尾松（Pinus massoniana）磷酸甘油酸激酶1（PGK1）与胞质溶胶葡萄糖磷酸异构酶（GPIC）的功能，采用RACE技术克隆了PmPGK1和PmGPIC基因，并进行了生物信息学分析与亚细胞定位，采用实时荧光定量PCR技术分析PmPGK1和PmGPIC的表达特性。结果表明，PmPGK1和PmGPIC全长为2 106和1 848 bp，分别编码507和566个氨基酸。PmPGK1和PmGPIC分别定位于叶绿体和胞质溶胶。PmPGK1表达量为新叶 > 老叶 > 新茎 > 根 > 花；而PmGPIC为老叶 > 花 > 新叶 > 新茎 > 根。低温胁迫24 h，PmPGK1和PmGPIC的表达量均随时间延长先降低后升高，且PmGPIC的表达量在处理2 h后即降至较低水平；高浓度CO₂胁迫24 h，PmPGK1的表达量随时间延长呈降低-升高-再降低的变化趋势，PmGPIC的表达下调但变化较不显著。因此，推测PmPGK1主要参与卡尔文循环及叶绿体/质体糖酵解，PmGPIC主要参与细胞质基质糖酵解；PmPGK1、PmGPIC活性在低温胁迫下均受抑制；PmPGK1活性在CO₂胁迫下受到显著抑制，而PmGPIC活性的影响不大。     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

Co-regulation with genes of phospholipid biosynthesis of the CTR/HNM1-encoded choline/nitrogen mustard permease in Saccbaromyces cerevisiae

An 815 by region of the promoter of the Saccharomyces cerevisiae gene CTR/HNM1, encoding choline permease was sequenced and its regulatory function analysed by deletion studies in an in-frame promoter-lacZ construct. In addition to the TATA box, a 10 by motif (consensus 5-CATGTGAAAT-3) was found to be mandatory for CTR/HNM1 expression. This decamer motif is located between nucleotides –262 and –271 and is identical in 9 of 10 by with the regulatory motif found in the S. cerevisiae INO1 and CHO1 genes. Constructs with the 10 by sequence show high constitutive expression, while elimination or alterations at three nucleotide positions, of the decamer motif in the context of an otherwise unchanged promoter leads to total loss of -galactosidase production. Expression of the CTR/HNM1 gene in wild-type cells is regulated by the phospholipid precursors inositol and choline; no such influence is seen in cells bearing mutations in the phospholipid regulatory genes INO2, INO4, and OPI1. There is no regulation by INO2 and OPI1 in the absence of the decamer motif. However constructs not containing this sequence (promoter intact to positions –213 or –152) are still controlled by INO4. Other substrates of the choline permease, i.e. ethanolamine, nitrogen mustard and nitrogen half mustard do not regulate expression of CTR/HNM1.     

副溶血弧菌VP2918基因缺失株的构建及功能研究

[目的] 以副溶血弧菌VP2918为研究对象,研究其对副溶血弧菌的生物学特性和致病性的影响。[方法] 利用同源重组技术构建了vp2918基因的基因缺失株（Δvp2918）和互补株（CΔvp2918）,并对野生株、缺失株和互补株的细菌生长曲线、运动性、生物被膜形成能力、对HeLa细胞的黏附能力、细胞毒性、对小鼠的致死率和组织载菌量进行分析。[结果] 缺失vp2918基因不影响副溶血弧菌的生长特性、运动性、生物被膜形成能力以及对HeLa细胞的黏附能力。但与野生株相比,Δvp2918对HeLa细胞的毒性作用显著降低;感染Δvp2918的小鼠症状明显减轻,存活率更高;Δvp2918在小鼠脾脏和肝脏中的载菌量显著低于野生株,互补株毒力基本恢复至野生株水平。[结论] vp2918不参与副溶血弧菌的运动性和生物被膜形成能力等过程,但与该菌的致病性相关,为潜在的毒力因子。