枯草芽孢杆菌生物学特性及其与稻瘟病菌互作的蛋白质组学研究

枯草芽孢杆菌（Bacillus subtilis）是革兰氏阳性细菌研究的模式菌株，为重要的生防菌剂。本论文以枯草芽孢杆菌ATCC6051作对照，首次对新型菌株KB-1111和KB-1122进行了生物学特性、生防潜力以及与水稻稻瘟病致病菌互作的蛋白质组学研究。 形态学观察表明，菌株KB-1111和KB-1122在细胞形态、芽孢的大小、运动性、菌落褶皱和色素的生成等方面与菌株ATCC6051相似，具有枯草芽孢杆菌的典型特征。生理生化测定以及对多种碳源的利用结果显示，三个菌株大部分指标检测结果相同，只在几个方面等存在差异。 体外平板对峙抑菌试验说明，枯草芽孢杆菌ATCC6051、KB-1111和KB-1122对8种作物、果蔬代表性病害致病真菌具有明显的拮抗效果。其中，菌株KB-1122的广谱抗真菌活性优于KB-1111，而KB-1111又强于对照菌株ATCC6051，尤其是对稻瘟病（M. grisea P131）和蔬菜菌核病（S. sclerotiorum）显现出强烈的抑制作用，具备生防拮抗菌的优秀性能。 比较蛋白质组学分析结果表明，液体悬浮培养枯草芽孢杆菌KB-1111、KB-1122二维蛋白质组表达谱至少有11个胞内蛋白和10个胞外蛋白出现丰度差异。其中，菌株KB-1122中胁迫或逆境反应相关ATP酶、顺乌头酸水合酶和alpha-淀粉酶前体在细胞内蛋白质组，以及分泌型蛋白―内切葡聚糖酶在胞外蛋白质组中的高丰度表达可能与菌株KB-1122的优势拮抗能力相关。 将对数生长期的枯草芽孢杆菌KB-1122与菌丝丰富期的稻瘟病菌P131悬浮混合共培养发现，在24小时的共培养过程中，稻瘟病菌P131菌丝体及芽管经历了致变、破裂、细胞质溢出直至菌丝体崩溃等一系列变化，枯草芽孢杆菌KB-1122表现出强烈的拮抗效应。差异显示蛋白质组学研究表明，共培养菌体蛋白质组至少有39个蛋白点丰度发生显著变化，其中33个蛋白点得到成功鉴定，包括12个上调蛋白和21个下调蛋白。根据鉴定结果分析，这些上调的蛋白质全部来源于枯草芽孢杆菌，而下调的蛋白全部属于稻瘟病菌。共培养过程中的培养液蛋白质组至少有20个蛋白点丰度发生显著变化，其中18个蛋白点得到成功鉴定。根据以上分析结果初步认为，3-磷酸甘油醛脱氢酶、丝氨酸蛋白激酶和内切葡聚糖酶在枯草芽孢杆菌KB-1122与稻瘟病菌P131相互作用的过程中可能是B. subtilis KB-1122发挥抗真菌活性的关键性蛋白。

Novel cathelicidin-derived antimicrobial peptides from Equus asinus

In the present study, EA-CATH1 and EA-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested PCR-based cloning strategy. Composed of 25 and 26 residues, respectively, EA-CATH1 and EA-CATH2 are smaller than most other cathelicidins and have no sequence homology to other cathelicidins identified to date. Chemically synthesized EA-CATH1 exerted potent antimicrobial activity against most of the 32 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, and minimal inhibitory concentration values against Gram-positive bacteria were mostly in the range of 0.3-2.4 mu g center dot mL-1. EA-CATH1 showed an extraordinary serum stability and no haemolytic activity against human erythrocytes in a dose up to 20 mu g center dot mL-1. CD spectra showed that EA-CATH1 mainly adopts an alpha-helical conformation in a 50% trifluoroethanol/water solution, but a random coil in aqueous solution. Scanning electron microscope observations of Staphylococcus aureus (ATCC2592) treated with EA-CATH1 demonstrated that EA-CATH could cause rapid disruption of the bacterial membrane, and in turn lead to cell lysis. This might explain the much faster killing kinetics of EA-CATH1 than conventional antibiotics revealed by killing kinetics data. In the presence of CaCl2, EA-CATH1 exerted haemagglutination activity, which might potentiate an inhibition against the bacterial polyprotein interaction with the host erythrocyte surface, thereby possibly restricting bacterial colonization and spread.

Frog skins keep redox homeostasis by antioxidant peptides with rapid radical scavenging ability

The question of how amphibians can protect themselves from reactive oxygen species when exposed to the sun in an oxygen-rich atmosphere is important and interesting, not only from an evolutionary viewpoint, but also as a primer for researchers interested in mammalian skin biology, in which such peptide systems for antioxidant defense are not well studied. The identification of an antioxidant peptide named antioxidin-RL from frog (Odorrana livida) skin in this report supports the idea that a peptide antioxidant system may be a widespread antioxidant strategy among amphibian skins. Its ability to eliminate most of the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical tested within 2 s, which is much faster than the commercial antioxidant factor butylated hydroxytoluene, suggests that it has a potentially large impact on redox homeostasis in amphibian skins. Cys10 is proven to be responsible for its rapid radical scavenging function and tyrosines take part in the binding of antioxidin-RL to radicals according to our nuclear magnetic resonance assay. (C) 2010 Elsevier Inc. All rights reserved.

A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae

Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 run. An optimized procedure yielded a high correlation coefficient (R-2 = 0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 mu g/mL and 20 mu g/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 mu g/mL and 2.0 mu g/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production. (C) 2009 Published by Elsevier B.V.

Comparative studies on physiological responses to phosphorus in two phenotypes of bloom-forming Microcystis

Toxic Microcystis blooms frequently occur in eutrophic water bodies and exist in the form of colonial and unicellular cells. In order to understand the mechanism of Microcystis dominance in freshwater bodies, the physiological and biochemical responses of unicellular ( 4 strains) and colonial ( 4 strains) Microcystis strains to phosphorus ( P) were comparatively studied. The two phenotype strains exhibit physiological differences mainly in terms of their response to low P concentrations. The growth of four unicellular and one small colonial Microcystis strain was significantly inhibited at a P concentration of 0.2 mg l - 1; however, that of the large colonial Microcystis strains was not inhibited. The results of phosphate uptake experiments conducted using P- starved cells indicated that the colonial strains had a higher affinity for low levels of P. The unicellular strains consumed more P than the colonial strains. Alkaline phosphatase activity in the unicellular strains was significantly induced by low P concentrations. Under P- limited conditions, the oxygen evolution rate, Fv/ Fm, and ETRmax were lower in unicellular strains than in colonial strains. These findings may shed light on the mechanism by which colonial Microcystis strains have an advantage with regard to dominance and persistence in fluctuating P conditions.

Strain Effects on the Optical Polarization Properties of R-Plane Wurtzite GaN

Using the effective-mass Hamiltonian for an arbitrary direction wurtzite semiconductor on the basis of k.p theory, we investigate the strain effects on the transition energies and optical properties in the R-plane ([1012]-oriented plane) GaN. The results show that (1) the transition energies decrease with the biaxial strains changing from -0.5 to 0.5%; and (2) giant optical anisotropy appears in the R-plane which is significantly affected by the biaxial strains. We clarify the relation between the strains and the polarization properties. Finally, we discuss the application of these properties to the R-plane GaN based devices. (c) 2009 The Japan Society of Applied Physics

INPLANE X-RAY-SCATTERING OF EPITAXIAL STRUCTURES

A new approach for in-plane X-ray scattering from the cleavages of epitaxial films or superlattices, where the scattering vectors are parallel to the interfaces, is proposed. This method can be employed to determine directly the in-plane X-ray strains and other atomic registry along the interfaces of the epitaxial structures.

染料脱色菌株筛选及染料脱色作用研究

染料废水成分复杂，色度高，是主要的环境污染源之一。应用微生物对染料废水脱色具有处理费用低和操作管理方便等优势，具有较好的发展前景。 本文采用筛选到一株短刺小克银汉霉(Cuuninghamella echinulata) KM3及一株红酵母(Rhodotorula sp.)YR-1分别对偶氮染料刚果红废水和酸性红废水进行了脱色研究，同时利用KM3及YR-1组成的复合菌种脱色体系对酸性红废水进行了脱色实验，为染料脱色真菌的应用奠定了基础。 KM3形成的菌丝球对多种染料具有较好脱色效果。KM3最适菌丝球形成条件：pH6-7、装液量90ml、孢子悬液浓度105个/ml (接种5ml)、摇床转速120rpm、培养温度30℃、培养时间72h；成球机理为凝聚型发育。KM3菌丝球对刚果红最佳脱色条件为：染料溶液初始pH值7.0，温度为33℃，摇床转速为120rpm。在刚果红50~200mg/l 浓度范围内， KM3菌丝球对刚果红吸附脱色动力学可用拟二级动力学方程描述。菌丝球对刚果红的吸附行为符合Langmuir和Freundlich 等温方程模型，Langmuir方程能更好的描述菌丝球对刚果红的吸附行为。丙酮对刚果红的解吸率最高，其解吸动力学过程符合二级动力学方程。将KM3应用在真菌生物滤床反应器中，在非灭菌和灭菌情况下接种孢子悬液后处理刚果红废水的过程中，均需长时间挂膜，处理效果一般。反应器中投加菌丝球后处理刚果红废水在3小时内达到92%以上的脱色效果。 YR-1对酸性红脱色机制属于降解脱色。YR-1对酸性红的最佳脱色培养条件为：初始pH 5.0、接种量5%、温度30℃；最佳碳源是葡萄糖，其次是蔗糖；最佳氮源是酵母膏或蛋白胨。在替换分批脱色实验和连续补料脱色实验中，脱色率一直保持在75%以上，显示YR-1在上述脱色体系中均具有良好适应性。 在三种复合菌种对酸性红的脱色体系中，体系Ⅲ（菌丝球与海藻酸钠固定化的酵母细胞混合培养）的脱色效果可达98%以上。对该体系而言，3种粒径固定化酵母小球（Ø1.1mm、1.8mm、3.1mm）中Ø1.8mm、3.1mm小球脱色率较为理想，而酵母细胞初始密度对脱色效果的影响不大。 KM3、YR-1分别具有处理刚果红废水和酸性红废水的应用前景，而KM3与YR-1联合应用对酸性红的脱色效果更好。

硅酸盐细菌的优选和发酵培养基的改进

硅酸盐细菌具有分解含钾矿物释放速效钾和产生植物生长刺激物质的作用，以硅酸盐细菌芽孢制成的硅酸盐细菌菌剂是生物肥料的一类重要产品.菌种性能、芽孢浓度和芽孢的存活能力是影响硅酸盐细菌菌剂质量的主要因素.该课题通过考察硅酸盐细菌菌株的适应性，辅以解钾活性综合评价了五株生产菌株的性能.研究探讨了芽孢存活能力与芽孢大小、芽孢浓度的关系，芽孢大存活能力强，芽孢浓度低易获得较大的芽孢.优化了原生产培养基，芽孢浓度稳定提高了4倍以上，芽孢形成率达98%，芽孢大小适中，生长时间、芽孢浓度稳定提高了4倍以上，芽孢形成率达98%，芽孢大小适中，生长时间、芽孢形成时间、发酵液pH和粘度均达到生产要求.同时对K<'+>对硅酸盐细菌生长的影响进行了研究，结果表明低浓度的K<'+>（约1×10<'-3>mol/L）对硅酸盐细菌的生长是有利的.

Enhanced Thermal Shock Resistance of Ceramics through Biomimetically Inspired Nanofins

We propose here a new method to make ceramics insensitive to thermal shock up to their melting temperature. In this method the surface of ceramics was biomimetically roughened into nanofinned surface that creates a thin air layer enveloping the surface of the ceramics during quenching. This air layer increases the heat transfer resistance of the surface of the ceramics by about 10 000 times so that the strong thermal gradient and stresses produced by the steep temperature difference in thermal shock did not occur both on the actual surface and in the interior of the ceramics. This method effectively extends the applications of existing ceramics in the extreme thermal environments.

Interface delamination of the thermal barrier coating subjected to local heating

To investigate the possible failure modes of the thermal barrier coating (TBC) used to protect the scramjet combustion chamber, the local heating via laser beam irradiation was utilized to simulate the service condition of high thermal flux and high temperature gradient. Firstly, the experimental method and process were described and the typical fracture morphology of the TBC under test were provided. Then, the theoretical and finite element modeling were carried out to study the temperature, deformation and stresses of the specimen when the top ceramic coat was subjected to local heating, and to demonstrate the mechanism on the failure of the TBC. It is revealed that the interface delamination shall appear and ultimately lead to the failure of the TBC under such thermal loading of local quick heating. According to the outcome of this study, the driving force of the interface delamination is influenced greatly by the key structural parameters and performance matching. Moreover, by utilizing the rules of the effects of these parameters on the fracture driving force, there is some possibility for the designer to optimize the performances of the TBC.

绞股蓝离体培养中影响皂甙产量的几个因素研究

绞股蓝(Gynostemma pentaphyllum)系葫芦科绞股蓝属植物，药用价值广泛，但其野生资源日趋减少，绞股蓝主要药效成分为绞股 蓝皂甙。利用组织和细胞培养生产绞股蓝皂甙是合理开发利用和保护绞股蓝资源的可能途径之一。本文对绞股蓝组织培养中培养基 的蔗糖和激素的组成以及各种胁迫条件：渗透压、重金属离子、真菌诱导物等对皂苷产量的影响进行了初步研究。其中，渗透压、 重金属离子、真菌诱导物对绞股蓝愈伤组织皂甙产量的影响尚未见报道。1. 蔗糖对绞股蓝愈伤组织之生长影响显著，2,4-D对绞股 蓝愈伤组织皂甙含量、产量影响显著。增加蔗糖用量，减少2，4-D的用量可提高皂甙产量。2. Mn++ 用量的提高抑制绞股蓝愈伤组 织的生长，但可促进皂甙含量、产量的提高。Mn++用量提高至MS培养基的20-30倍时可使皂甙产量增加近一倍，而提高Cu++浓度的 作用不明显。3. 甘露醇用量增加抑制绞股蓝愈伤组织的生长，但可使皂甙含量、产量提高。0.680mol·l-1甘露醇可使皂甙产量提 高83%，而Nacl较大抑制愈伤组织的生长并使皂甙产量降低。4. 米曲霉粗提物对绞股蓝愈伤组织生长先略微促进，然后抑制，而根 霉粗提物则使愈伤组织生长受抑制；两者对皂甙含量、产量的作用相似：在较低浓度范围内升高，然后下降。米曲霉粗提物可提高 产量一倍，根霉粗提物可提高42%。这些结果为高产细胞系的筛选和生长、生产培养条件的优化积累了资料。在综述部分，对植物 细胞培养中组织和器官分化、细胞结构变化、生化水平的变化与次生物合成和积累的关系作了讨论。Gynostemma pentaphyllum blongs to Gynostemma, Cucurbitaccae. It has a wide medical use, but its wild resource is threatened by people's excessive use. Its effective medical components are gypenosides. For reasonable use and protect its resource, it is a possible way to product gypenosides by plant tissue and cell culture. This paper has a primary study on the components of sucrose and hormones and a variety of stress conditions: osmostic pressure, heavy metal ion, fungal elicitors in the medium for the calli culture. The effects of osmostic pressure, heavy metal ion and fungal elicitors on the calli of Gynostemma pentaphyllum have not been reported. 1. Sucrose had a significant effect on the growth of the calli, 2,4 D had notable effects on the gypenosides content and production of the calli. Increased the concentration of sucrose and decreased the concentration of 2,4 D improved the production of gypenosides. 2. Increased the concentration of Mn++ inhibited the growth of the calli, but improved the content and production of gypenosides. The optimum concentration was 20-30 times as MS medium which improved the production 100%. Increased the concentration of Cu++ had not a notable effect. 3. Increased the concentration of mannitol inhibited the growth of the calli, but improved the content and production of gypenosides. The optimum concentration was 0.680mol·l-1 which improved the production 83%. Nacl apparently inhibited the growth of the calli and decreased the production of gypenosides. 4. The crude preparation of Aspergillus oryzae inhibited the growth of the calli that in low concentration. The crude praparation of Rhizopus formosensis inhibited the growth of the calli throughout. Their effects on the content and production of gypenosides are alike, but the former is higher than the latter. On the optimum concentration, each crude preparation improved the production 100% (Aspergillus oryzae), 42%(Rhizopus formosensis). These results has accumulated some informantion on the select of high yield cell strains and choose the best culture conditons for the growth and gypenosides product of the calli. In the review, it is discussed that the differentiation on tissue-organal, cellular and biochemical levels related to the synthesis and accumulation of secondary metabolites in plant culuture.

两株耐碱性木聚糖酶产生菌的筛选及产酶条件研究

本文主要研究了从造纸厂碱性土壤中筛选得到的，能够产生耐碱木聚糖酶的两株放线菌X24-14和X15-17。通过16 S rRNA基因序列分析并结合菌株的形态特征以及生理生化特性，初步认为菌株X15-17为拟诺卡氏菌属（Nocardiopsis）的一个潜在新种；菌株X24-14为纤维化纤维菌（Cellulosimicrobium cellulans）。 在此基础上探索了菌株X24-14和菌株X15-17所产木聚糖酶的基本酶学性质。研究发现，两株菌所产的木聚糖酶的耐碱性均较强： 1）菌株X24-14所产的木聚糖酶，在pH 4.2～9.4的范围内能维持较高的活力，pH 9.4条件下，仍能保持80％的酶活力；2）菌株X15-17所产的木聚糖酶在pH 4.0～9.0的范围内能维持较高的活力，pH 9.0条件下，仍能保持80％的酶活力；3）两株菌所产的木聚糖酶均具有较好的pH稳定性，在pH 2.0～11.0范围内稳定，pH 11.0、4 ℃条件下处理24 h仍具有75％的活力。 本文还重点研究了菌株X24-14在不同培养基成分及不同培养条件下的产酶情况，确定了其适宜的产酶条件。结果显示，菌株X24-14的最适碳源为麸皮；最适氮源为蛋白胨；最适产酶pH为pH 8.5。菌株X24-14适宜的产酶条件为：麸皮60 g/L，蛋白胨10 g/L，K2HPO4 7.0 g/L，pH 8.5，接种量为5％，37 ℃，200 r/min发酵培养108 h。 Two strains of actinomycetes, X24-14 and X15-17, which produced alkali-tolerant xylanase were screened from the soil samples collected from a pulp mill in china. Based on the morphological, physiochemical characteristics and 16S rRNA sequence, X24-14 was priminarily identified as cellulosimicrobium cellulans ; X15-17 was priminarily identified as a new species of Nocardiopsis. The investigation examined the enzyme activities which produced by X24-14 and X15-17 under different pH and different temperatures. The results showed that : 1）The xylanase from X24-14 had characteristic of alkali-tolerance: It remains 80% relative activity at pH ranges between pH 4.2 and pH 9.4 under 50℃. 2)The xylanase from X15-17 also showed characteristic of alkali-tolerance, it remains 80% relative activity at pH ranges between pH 4.0and pH 9.0 under 50℃. 3）The xylanase from the two strains showed alkali-stable characteristics. They were stable at pH ranges between pH 2.0 and pH 11.0, showing 75% of its maximal activity remaining under 24 hours of treatment at 4℃. We also studied the effect of different growth conditions: carbon source, nitrogen sources, inoculum size, and initial pH on the production of xylanase of strain X24-14. The results showed that :The optimal carbon source was wheat bran; The optima nitrogen source was peptone; The maximum xylanase activity was achieved in the medium containing 60 g/L wheat bran, 10 g/L peptone, 7 g/L K2HPO4, inoculum size 5% and pH 8.5, under 37℃ in 108 h.

Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.