类产碱假单胞菌杀虫蛋白的N端鉴定及抗体制备

The insecticidal protein from Pseudomonas pseudoaligenes was and exotoxin which had toxicity on locusts.In order to elucidate its molecular properties an amino acid sequence,the insecticidal protein was purified from the culture supernatant by ultrafiltration,ion\|exchange chromatography and gel filtration,and showed a single band on SDS\|PAGE.Analysis of the purified insecticidal protein dentified N\|terminal sequence of ten amino acid residues.Its polyclonal antibody was also obtained by immunizing rabb…     

猪苓分生孢子产生的电镜研究

The ultrastructure of conidia produced by the mycelia of Polyporus umbellatus was studied by use of scanning and transmission electron microscopy. The results suggested that only clamped (dikaryotic) mycelia produced aerial conidiophores with ovoid to rod-shaped conidia. There was a distinctive bulge near the top of the conidium. The contents of the conidia were dense. This type of conidium could be regarded as arthroconidia.     

产苯乙醇胺醇脱氢酶菌种筛选与发酵条件研究

A Arachnia sp.P163 producing alcohol dehydrogenase which is able to reduce aminoacetophenone to R\|1\|phenyl\|2\|aminoethanol was obtained from soil and cultures.The maximum activity of enzyme was produced by the LB medium containing 1% sodium citrate and peptone,0 1% phenylaminoethanol as inducer at 30℃ for 48 hs.     

Purification and properties of glutamate synthase from Streptomyces lincolnensis

Glutamate synthase (EC 1 4 1 14) was purified to homogeneity from a cell\|free extract of Streptomyces lincolnensis by precipitation with streptomycin sulfate and ammonium sulfate, and column chromatography on DEAE\| cellulose, Sepharose 6B, DEAE\|sephadex A\|50, hydroxyapatite and Sephadex G\|150. The enzyme activity is stabilized by addition of α ketoglutarate, PMSF,EDTA, β mercaptoethanol and glycerol. The native enzyme has a molecular weight of 138 000 and is composed of two nonidentical subunits with molecular weights of 81 000 and 57 000. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The enzyme shows optimum activity at pH 7.2 and 30℃. Km values for α ketoglutarate, L\|glutamine and NADH were 417, 435, and 52.1 μmol/L, respectively. When NADPH was substituted for NADH as reductant, there was approximately 13% of the control activity. The activity of this glutamate synthase is inhibited by its products (i.e. glutamate and NAD), several metal ions, amino acids and tricarboxylic acid cycle intermediates.     

Optimization of PCR protocol in microsatellite analysis with silver and SYBR® stains

Here we present the optimization of PCR conditions for microsatellite analysis of coniferous trees. The use of touchdown protocol for annealing resulted in a high success rate for optimization using fewer temperature profiles. The use of SYBR^￠ Green gel stain to detect PCR products in agarose gels was more sensitive than ethidium bromide. This is valuable for determining the success of PCR reactions and estimating the amount of PCR products formed—which is crucial in determining the dilution required to produce bands of similar intensity upon silver staining of the polyacrylamide gels. The use of SYBR^￠ Gold for staining polyacrylamide gels was not satisfactory in terms of the image quality produced. However, it was comparable to silver staining in terms of sensitivity, and could possibly be used in cases where the products are present as sharp single bands. In those cases, the use of SYBR^￠ Gold gel stain would save time and money for staining polyacrylamide gels.     

转基因烟草中的海藻糖测定

The \%E.coli\% trehase synthalose gene(\%otsA\%) was transferred into \%Nicotiana tabacum\% mediated by \%Agrobacterium\%, but the method for detecting low concentration of trehalose in transgenic plant was not available.The high performance liquid chromatograph(HPLC) with evaporative light\|scatting detector (ELSD) using water:methyl cyanide(1∶2\^6 v/v) as mobile phase was established in this work. An ODS column Zorbax RX\|SIL was employed. The trehalose detection limits of ELSD was 5mg/L. From the linear…     

Properties of serine：glyoxylate aminotransferase purified from Arabidopsis thaliana leaves

The photorespiratory enzyme L-serine：glyoxylate amino- transferase （SGAT; EC 2.6.1.45） was purified from Arabidopsis thaliana leaves. The f＇mal enzyme was approximately 80 % pure as revealed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis with silver staining. The identity of the enzyme was confirmed by LC/MS/MS analysis. The molecular mass estimated by gel filtration chromato- graphy on Sephadex G-150 under non-denaturing conditions, mass spectrometry （matrix-assisted laser desorption/ ionization/time of flight technique） and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82.4 kDa, 42.0 kDa, and 39.8 kDa, respectively, indicating dimer as the active form. The optimum pH value was 9.2. The enzyme activity was inhibited by aminooxyacetate and β-chloro-L-alanine both compounds reacting with the carbonyl group of pyridoxal phosphate. The enzyme＇s transaminating activity with L-alanine and glyoxylate as substrates was approximately 55 % of that observed with L-serine and glyoxylate. The lower Kmvalue （1.25 mM） for L-alanine, compared with that of other plant SGATs, and the kcat/Km（Ala） ratio being approxi- mately 2-fold higher than kcat/Km（Ser） suggested that, during photorespiration, Ala and Ser are used by Arabidopsis SGAT with equal efficiency as amino group donors for glyoxylate. The equilibrium constant （Keq）, derived from the Haldane relation, for the transamination reaction between L-serine and glyoxylate with the formation of hydroxypyruvate and glycine was 79.1, strongly favoring glycine synthesis. However, it was accompanied by a low Km value of 2.83 mM for glycine. A comparison of some kinetic properties of the studied enzymes with the recombinant Arabidopsis SGATs previously obtained revealed substantial differences. The ratio of the velocity of the transamination reaction with L-alanine and glyoxylate as substrates versus that with L-serine and glyoxylate was 1：1.8 for the native enzyme, whereas it was 1：7 for the recombinant SGAT. Native SGAT showed a much lower Km value for L-alanine compared to the recombinant enzyme.     

前β_1-HDL与细胞HDL受体结合活性的研究

The activities of pre\|β 1HDL and apoE\|deficient HDL 3 binding to HDL receptors on human SMC,HepG2 and macrophage were detected using enzyme linked immune receptor assay(ELIRA).The results showed the value of K d of pre\|β 1 HDL binding to HDL receptor was significantly lower than that of apoE\|deficient HDL 3( P <0.05),but B max showed no difference.The results suggested that the affinity of pre\|β 1 HDL to HDL receptor was significantly higher than that of apoE\|deficient HDL 3.     

蝉拟青霉代谢产物清除DPPH自由基和抗真菌活性的研究

DPPH-TLC and DPPH-Microplate assays were used to determine the free radical scavenging activities of methanol extracts from the fermentation broth and the mycelia of Paecilomyces cicadae P3. The results revealed that the extracts had strong free radical scavenging activities. At the concentration of 5.0mg/mL the methanol extract from fermentation broth of P3 could decrease 74.86% of 0.4mg/mL DPPH radicals after incubating at 37℃ for 10 minutes. An anti-fungal TLC assay was applied to test antimicrobial metabolites of Paecilomyces cicadae P3. Meanwhile, an anti-Candida albicans Oxford Cup assay was made to confirm their anti-fungal activities. At the concentration of 5.0mg/mL the methanol extracts from fermentation broth and mycelia of P3 had significant anti-Candida albicans activities and the diameters of inhibition zone were 21.42mm and 11.23mm respectively.     

简单快速的DNA银染和胶保存方法

本文介绍了一套简单快速的DNA银染以及胶保存的方法,整个过程仅需10~15分钟,而且背景浅,条带清楚,灵敏度高,稳定性好。胶保存采用双层玻璃纸夹心法,可长久地保存胶显色时的原貌。以常规PAG胶检测和HLA的SSCP分型为例,利用该套方法进行了银染以及胶的保存,均得到了满意的结果。该方法具有推广价值。 Abstract:This paper introduced the simple and rapid methods of silver staining and gel preservation.It was taken only about 10 and 15 minutes to stain a gel.The background of gel was light,the bands were clear,the sensibility was high and the stabilization was well by the method of silver staining.The gel preservation adopted a method named two-layer transparent plastic paper "Sandwich" which could keep the gel with primitive colors for a long time.The methods were used on PAG checking and SSCP typing of HLA and the results were satisfactory.The set of methods are expected to be widely used in laboratories.     

飞虱虫疠霉液培菌丝的凝胶化及其杀蚜活性*

用丙烯酰胺淀粉凝胶包埋飞虱虫疠霉(Pandora delphacis)液培菌丝,制成含营养和不含营养的虫霉凝胶,并测试其产孢能力及对桃蚜(Myzus persicae)的侵染力。经持续9 d产孢观察,相同菌丝量条件下,含营养的虫霉凝胶的累计产孢量(1910.9 个/mm2）为不含营养的虫霉凝胶的3.14倍（608.5个/mm2）和对照（纯液培菌丝）的4.69倍（387.5个/mm2）。用虫霉凝胶的“孢子浴”接种桃蚜居群（成蚜与不同龄期若蚜的混合）,在2.23个/mm2的剂量下,接种后第7 d的死亡率达82.3%。结果表明,丙烯酰胺淀粉凝胶作为载体材料,与飞虱虫疠霉的基本生物学特性相容,对虫霉的剂型化具有重要意义     

扫描电镜观察飞虱虫疠霉侵染水稻害虫褐飞虱(英文)

稻飞虱如褐飞虱频繁威胁东亚和南亚的水稻生产。化学杀虫剂作为控制虫害的主要手段时常引发环境问题和害虫抗药性。昆虫病原真菌飞虱虫疠霉是稻飞虱的天敌,了解该菌的生物学和流行学特性是开发其生物防治潜能的必要前提。研究利用扫描电镜对飞虱虫疠霉侵染寄主褐飞虱的关键步骤进行了观察。飞虱虫疠霉的分生孢子接触到褐飞虱体壁后可迅速萌发产生侵染性芽管,侵入虫体。经过4d左右的体内潜伏期,飞虱虫疠霉菌丝重新在寄主体表出现。最先突破坚硬体壁的部位为虫体腹部。3种特化菌丝形成于虫体表面,包括假囊状体、假根和分生孢子梗。假囊状体向虫体四周空气中伸出,很可能是帮助真菌探知周围空气湿度状况并吸收湿气用于开启新一轮的侵染循环。假根将虫尸固定在原位植株上,利于之后分生孢子梗主动弹射侵染性分生孢子感染附近健康寄主。这次观察结果显示飞虱虫疠霉具备用于稻飞虱生防的潜能,有必要开展其扩大培养和剂型化研究推进该生防菌的田间应用。     

Sporulation,storage and infectivity of obligate aphid pathogen Pandora nouryi grown on novel granules of broomcorn millet and polymer gel

Aims: Producing granular cultures of obligate aphid pathogen Pandora nouryi for improved sporulation and storage. Methods and Results: Small millet–gel granules were made of the mixtures of 80–95% millet powder with 5–20% polymer gel (polyacrylamide, polyacrylate or acrylate‐acrylamide copolymer) and inoculated with mycelia at 30 mg biomass g^?1 dry granules plus 87·5% water, followed by static incubation at 20°C for 4–12 days. The fungus grew well on 12 preparations but best on that including 10% copolymer. An 8‐day culture of this preparation discharged maximally 58·5 × 10⁴ conidia mg^?1 granule at 100% RH and was capable of ejecting conidia at the nonsaturated regimes of 86–97% RH. During storage at 6°C, granular cultures with >85% water content had twofold longevity (120 days) and half‐decline period (34–36 days) of those stored at room temperature. The steadily high water content preserved the cultures better than that decreasing at 6°C. However, conidia from 70‐day‐stored granules were less infective to Myzus persicae nymphs than those from fresh ones based on their LC₅₀s. Conclusions: The millet–gel granules had higher sporulation capacity than reported Pandora cultures and a capability of spore discharge at nonsaturated humidity. Significance and Impact of the Study: The granular cultures are more useful for aphid control.     

适合飞虱虫疠霉菌丝生产的液体培养基组分及发酵条件

蚜虫、飞虱、叶蝉等是威胁农业生产的刺吸式口器害虫,长期以来依赖化学农药防治,严重影响农产品的安全性和农业生态环境.探讨这类害虫的生物防治途径,减轻农产品化学农药的残留污染,一直受到国内外学者的关注.虫霉(En-tomophthorales)的许多种类具有优异的诱病杀虫能力,尤其通过主动弹射孢子而在害虫种群中高强度流行的特征,可在短期内迅速压低虫口密度,是极其重要的杀虫微生物资源[1～3].     

飞虱虫疠霉继发性感染对桃蚜数量增长的控制作用

用飞虱虫疠霉（Pandora delphaics)“孢子浴”接种的桃蚜(Myzus persicae)无翅成蚜在离体甘蓝菜叶片（65cm^2)上建立蚜群,在不同温度（10－30℃）和湿度（74％－100％RH）的组合条件下任其繁衍,发病和交互感染,以评价该菌的控蚜效果。在25个温,湿度组合处理（8次重复,每重复含3头接种成蚜)中,蚜群均不同程度的发病死亡,在历时30d的观察中,以高温（20－30℃）,高湿（95％RH）组合条件下的蚜群发病快且死亡率高,蚜尸上产生的孢子有效地引起若蚜继发性感染。与相同温度下不带菌的对照蚜群相比,次于30℃下,各湿度除个别例外,第8d的控蚜率达30％以上,第20d达80％以上。在10℃和15℃下,控蚜效果一般不如上述较高温度下,且与湿度的关联程度相对较低,但最大控蚜效果均发生在100％RH处理中。结果表明,飞虱虫疠霉用于蚜虫防治的潜力很大,值得深入研究和开发利用。     

光周期对飞虱虫疠霉离体培养的生物量及其产孢节律的影响

飞虱虫疠霉（Ｐａｎｄｏｒａｄｅｌｐｈａｃｉｓ）是侵染飞虱和叶蝉等重要作物害虫的昆虫病原真菌。通过在液体及平板培养中研究光周期对菌丝及菌落生长量、产孢节律和产孢量的影响,作者发现光照对液培菌丝的营养生长无显著影响,但长光照液培菌丝的产孢延迟并且产孢量减少,而全黑暗液培菌丝产孢早且产孢量大。但是,光照明显促进全黑暗下液培菌丝的产孢。在平板培养中,长光照能促进菌落生长和产孢;半光照半黑暗虽有利菌落生长,但产孢量很低;光照９ｈ和１８ｈ产孢量较大,但菌落生长较小。无论液体还是平板培养,飞虱虫疠霉均生长良好,但建议在全黑暗条件下进行液体培养,在长光照下进行平板培养。     

Effects of temperature and relative humidity on sporulation of Metarhizium anisopliae var. acridum in mycosed cadavers of Schistocerca gregaria.

The effects of relative humidity (RH) and temperature on the sporulation of Metarhizium anisopliae var. acridum on mycosed cadavers of desert locust, Schistocerca gregaria, were assessed in the laboratory. Quantitative assessments of conidial production over 10 days under constant conditions showed that sporulation was optimized at RH > 96% and at temperatures between 20 and 30 degrees C. Under both these conditions >10(9) conidia/cadaver were produced. At 25 degrees C, conidial yield was maximized under conditions in which cadavers remained in contact with damp substrate. Relatively little sporulation occurred at 15 degrees C (< 3 x 10(7) conidia/cadaver) and 40 degrees C (< 4 x 10(6) conidia/cadaver) and no sporulation occurred at 10 or 45 degrees C. Following incubation, conidial yield was closely related to the water content of locust cadavers. In separate tests, locust cadavers were incubated for 10 days under diurnally fluctuating temperature and RH that comprised favorable (25 degrees C/100% RH) alternating with unfavorable (40 degrees C/80% RH) conditions for sporulation. In this case, fewer conidia were produced compared with cadavers that were incubated under the favorable conditions for an equal period cumulatively but were not periodically exposed to unfavorable conditions. However, this reduced sporulation observed with the fluctuating condition was not observed when cadavers were similarly incubated under favorable/unfavorable conditions of temperature but were not periodically exposed to the low RH condition. This result implies that sporulation is a dynamic process, dependent not only on periodic exposure to favorable RH but also on the interrelation of this with low RH. Associated tests and the monitoring of changes in cadaver weights imply that the mechanism driving the reduced sporulation under fluctuating RH is the net water balance of cadavers, i.e. the cumulative ability of the fungus/cadaver to adsorb water necessary for sporulation at high RH is restricted by water loss associated with intermittent exposure to a low RH. The duration of daily exposure to high humidity appears to be a crucial constraint to the recycling ability of M. anisopliae var. acridum.     

植物油对虫霉菌液体培养与保存的作用*

在萨氏营养液（ＳＤＢ）中加入用 ０．１％蔗糖脂肪酸酯－１５（ＳＥ１５）乳化的０．５０％芝麻油,可使在ＳＤＢ中生长慢而结块的新蚜虫疠霉、安徽虫瘟霉、飞虱虫疠毒和根虫瘟霉等四种虫霉的五个菌株形成均匀分散的菌丝体和能稳定转接的菌液。在ＳＤＢ中加入乳化芝麻油 （ ０．２５％～１．５％）、菜籽油（ ０．５０％～１．５％）和色拉油（ ８０％花生油和 ２０％菜籽油,０．７５％～１．５％）,能明显提高新蚜虫疠霉Ｆ９８０１８的生物量;而在ＳＤＢ中加入０．５０％乳化芝麻油,可使液培菌丝产孢量增加近一倍;在ＳＤＢ中加入的三种植物油（０．７５％）,在Ｆ９８０１８生长４ｄ后,含量分别下降了８５％～９４％,但其中脂肪酸相对组成基本不变,表明该菌生长能充分利用植物油中各脂肪酸。用萨氏培养基（ ＳＤＡＹ）、加入 ０．５０％乳化芝麻油的 ＳＤＡＹ（ ＯＳ－ＳＤＡＹ）和牛奶蛋黄培养基（ＳＥＭＡ）培养安徽虫瘟霉Ｆ９７０２８和新蚜虫疠霉Ｆ９８０２８,两菌株在ＳＥＭＡ上生长最快;在ＯＳ－ＳＤＡＹ以较慢速度正常生长;而在ＳＤＡＹ上,两菌株或不能生长,或在第二次培养时出现明显退化。用ＳＥＭＡ和ＯＳ－ＳＤＡＹ在３℃下来保存上述四种虫霉的七个菌株,在ＯＳ－ＳＤＡＹ上?     

九州虫草菌丝体对Mn的耐性及富集

重金属耐性真菌的研究是生物修复的重要研究内容。本文研究了九州虫草（Cordyceps kyusyuensis）对于Mn的耐性及富集。在液体培养基中添加不同浓度（0—60 g/L）的Mn离子,测定其菌丝生物量、菌丝Mn含量、菌丝抗氧化酶活性和过氧化水平以及菌体细胞离子交换量、Mn在细胞中的分布的变化情况。实验结果表明九州虫草菌丝生物量与Mn浓度呈显著负相关,Mn浓度60 g/L为九州虫草菌丝生长极限浓度。菌丝中Mn含量随培养基中Mn浓度的增大而显著升高,10 g/L Mn时,菌丝细胞中Mn积累量达到细胞干重的1.0013%。九州虫草菌丝中过氧化产物丙二醛（MDA）、可溶性蛋白（SP）含量、可溶性糖浓度与培养基中Mn浓度呈负相关,实验组与对照组差异显著。抗氧化酶（过氧化氢酶（CAT）、过氧化物酶（POD）、超氧化物歧化酶（SOD））活性随着培养基中Mn浓度增大而显著升高,但变化趋势不同。九州虫草菌丝细胞不可溶性组分中Mn的量（91.51%—98.6%）显著高于可溶部分（1.40%—8.49%）。九州虫草菌丝细胞壁离子交换量（CEC）随着培养基中Mn浓度的升高变化不明显。说明在九州虫草菌丝对Mn的富集过程中,其细胞壁、细胞膜和细胞器对于Mn结合发挥了主要作用,细胞质中可溶性成分对Mn的结合发挥次要作用。在Mn的胁迫下,增强抗氧化酶系统的协同作用以清除大量自由基是细胞对锰耐性的重要机制。     

Gallotannin hydrolysis by immobilized fungal mycelia in a packed bed bioreactor.

Hydrolysis of gallotannin to gallic acid by immobilized mycelia of Aspergillus niger MTCC 282, Aspergillus fischerii MTCC 150, Fusarium solani MTCC 350 and Trichoderma viride MTCC 167 in a packed bed bioreactor was studied. Fungal mycelia preinduced with 5 g L-1 gallotannin were immobilized in calcium alginate gel (1.5%) and the resultant beads were packed in a column to a bed volume of 175 mm3. Gallotannin dissolved in distilled water was passed through the column and the eluate was recycled after adjusting pH to 6 with ammonium hydroxide (10%). Maximum hydrolysis of gallotannin was recorded by immobilized mycelia of F. solani and T. viride at 35 degrees and 45 degrees C after 175 and 60 min of residency period respectively. Optimum substrate concentration required for maximum hydrolysis was 10 g L-1 at pH 5 for both the fungi. Immobilized mycelia of A. niger and A. fischerii revealed maximum operational stability. Loss of activity after eighth run was in the order of-A. niger (no loss), A. fischerii (7.5%), F. solani (18%) and T. viride (18%). Stability in terms of retention of enzyme activity after 150 days of storage at 4 degrees C was A. niger (58%), A. fischerii (26.8%), F. solani (83%) and T. viride (85.1%).