Degradation of (+/-)-synephrine by Arthrobacter synephrinum. Oxidation of 3,4-dihydroxyphenylacetate to 2-hydroxy-5-carboxymethyl-muconate semialdehyde.

1. Cell-free extracts of Arthrobacter synephrinum catalyse the oxidation of 3,4-dihydroxy-phenylacetate. 2. The product of oxidation was characterized as 2-hydroxy-5-carboxymethylmuconate semialdehyde from its chemical behaviour as well as from nuclear-magnetic-resonance spectra. 3. A 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15) was partially purified from A. synephrinum. 4. The enzyme had a Km of 25 micrometer towards its substrate and exhibited typical Michaelis-Menten kinetics. 5. The enzyme also catalysed the oxidation of 3,4-dihydroxymandelate and 3,4-dihydroxyphenylpropionate, at reaction rates of 0.5 and 0.04 respectively of that for 3,4-dihydroxyphenylacetate. 6. The enzyme was sensitive to treatment with thiol-specific reagents. 7. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography was approx. 282000.

Studies on the metabolism of β-carotene and apo-β-carotenoids in rats and chickens

1. (1) The relative abilities of the various cell fractions of rat and chicken liver to oxidize and reduce retinal and 8'- and 12'-apo-β-carotenal were investigated and it has been shown that, while retinal is exclusively oxidized by the soluble fraction, the apocarotenals are mostly oxidized by the particulate fractions of the homogenate. 2. (2) Addition of NAD+ or NADP+ markedly activated the oxidation of the apocarotenals, but not of retinal by the particulate fractions. 3. (3) Considerable amounts of retinal and 8'-, 10'- and 12'-apo-β-carotenal were isolated from the intestine of chickens fed β-carotene and these apocarotenoids were conclusively identified. 4. (4) Significant amounts of 8'-, 10'- and 12'-apo-β-carotenoic acids were isolated from the intestine of rats given 8'-apo-β-carotenal and these apocarotenoic acids were also conclusively identified. 5. (5) In the light of these observations it is suggested that during conversion to vitamin A, the β-carotene molecule is simultaneously attacked by the dioxygenase at several double bonds, the primary attack being at the central double bond and a tentative scheme for the mechanism of conversion is proposed.

Plasmids and aromatic degradation in Sphingomonas for bioremediation : Aromatic ring cleavage genes in soil and rhizosphere

Microbial degradation pathways play a key role in the detoxification and the mineralization of polyaromatic hydrocarbons (PAHs), which are widespread pollutants in soil and constituents of petroleum hydrocarbons. In microbiology the aromatic degradation pathways are traditionally studied from single bacterial strains with capacity to degrade certain pollutant. In soil the degradation of aromatics is performed by a diverse community of micro-organisms. The aim of this thesis was to study biodegradation on different levels starting from a versatile aromatic degrader Sphingobium sp. HV3 and its megaplasmid, extending to revelation of diversity of key catabolic enzymes in the environment and finally studying birch rhizoremediation in PAH-polluted soil. To understand biodegradation of aromatics on bacterial species level, the aromatic degradation capacity of Sphingobium sp. HV3 and the role of the plasmid pSKY4, was studied. Toluene, m-xylene, biphenyl, fluorene, phenanthrene were detected as carbon and energy sources of the HV3 strain. Tn5 transposon mutagenesis linked the degradation capacity of toluene, m-xylene, biphenyl and naphthalene to the pSKY4 plasmid and qPCR expression analysis showed that plasmid extradiol dioxygenases genes (bphC and xylE) are inducted by phenanthrene, m-xylene and biphenyl whereas the 2,4-dichlorophenoxyacetic acid herbicide induced the chlorocatechol 1,2-dioxygenase gene (tfdC) from the ortho-pathway. A method to study upper meta-pathway extradiol dioxygenase gene diversity in soil was developed. The extradiol dioxygenases catalyse cleavage of the aromatic ring between a hydroxylated carbon and an adjacent non-hydroxylated carbon (meta-cleavage). A high diversity of extradiol dioxygenases were detected from polluted soils. The detected extradiol dioxygenases showed sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Five groups of extradiol dioxygenases contained sequences with no close homologues in the database, representing novel genes. In rhizoremediation experiment with birch (Betula pendula) treatment specific changes of extradiol dioxygenase communities were shown. PAH pollution changed the bulk soil extradiol dioxygenase community structure and birch rhizosphere contained a more diverse extradiol dioxygenase community than the bulk soil showing a rhizosphere effect. The degradation of pyrene in soil was enhanced with birch seedlings compared to soil without birch. The complete 280,923 kb nucleotide sequence of pSKY4 plasmid was determined. The open reading frames of pSKY4 were divided into putative conjugative transfer, aromatic degradation, replication/maintaining and transposition/integration function-encoding proteins. Aromatic degradation orfs shared high similarity to corresponding genes in pNL1, a plasmid from the deep subsurface strain Novosphingobium aromaticivorans F199. The plasmid backbones were considerably more divergent with lower similarity, which suggests that the aromatic pathway has functioned as a plasmid independent mobile genetic element. The functional diversity of microbial communities in soil is still largely unknown. Several novel clusters of extradiol dioxygenases representing catabolic bacteria, whose function, biodegradation pathways and phylogenetic position is not known were amplified with single primer pair from polluted soils. These extradiol dioxygenase communities were shown to change upon PAH pollution, which indicates that their hosts function in PAH biodegradation in soil. Although the degradation pathways of specific bacterial species are substantially better depicted than pathways in situ, the evolution of degradation pathways for the xenobiotic compounds is largely unknown. The pSKY4 plasmid contains aromatic degradation genes in putative mobile genetic element causing flexibility/instability to the pathway. The localisation of the aromatic biodegradation pathway in mobile genetic elements suggests that gene transfer and rearrangements are a competetive advantage for Sphingomonas bacteria in the environment.

The quest for golden bananas: Investigating carotenoid regulation in a Fe’i group Musa cultivar

The regulation of carotenoid biosynthesis in a high-carotenoid-accumulating Fe’i group Musa cultivar, “Asupina”, has been examined and compared to that of a low-carotenoid-accumulating cultivar, “Cavendish”, to understand the molecular basis underlying carotenogenesis during banana fruit development. Comparisons in the accumulation of carotenoid species, expression of isoprenoid genes, and product sequestration are reported. Key differences between the cultivars include greater carotenoid cleavage dioxygenase 4 (CCD4) expression in “Cavendish” and the conversion of amyloplasts to chromoplasts during fruit ripening in “Asupina”. Chromoplast development coincided with a reduction in dry matter content and fruit firmness. Chromoplasts were not observed in “Cavendish” fruits. Such information should provide important insights for future developments in the biofortification and breeding of banana.

Chemoenzymatic Synthesis of Chiral Organosulfur Compounds

This thesis is primarily concerned with the enzyme- catalysed synthesis of sulfoxides using reductase and dioxygenase enzymes. Chapter 1 provides an introduction to the topic of redox chemistry with particular emphasis on the application of reductase and dioxygenase enzymes in organosulfur chemistry. Earlier literature methods for the production of enantiopure sulfoxides are reviewed. A brief discussion of the methods used for the determination of enantiomeric excess and absolute configuration is provided. Chapter 2 contains results obtained using a range of whole-cell bacteria each using a dimethyl sulfoxide reductase enzyme. The synthesis of a series of racemic sulfoxides and the development of appropriate CSPHPLC analytical methods is discussed. Kinetic resolutions of a series of sulfoxides have been achieved. Chapter 3 contains a presentation of results using dioxygenase enzymes as biocatalysts for the asymmetric sulfoxidation of dialkyl sulfoxides including thioacetal sulfoxides. A new range of monosulfoxides, cis-dihydrodiols and cis- dihydrodiol sulfoxides have been isolated in enantiopure form. Chapter 4 is focussed on the application of chiral sulfoxides in synthesis. A new chemoenzymatic route to diol sulfoxide enantiomers and the derived enantiopure phenols and catechols is discussed. The application of chemically synthesised sulfoxide enantiomers in the production of hydroxy sulfoxides is reported. Chapter 5 provides a full experimental section where the synthesis of sulfides and racemic sulfoxides is included. The methods used in the isolation and characterisation of bioproducts from the biotransformation are discussed and full experimental details given.

New pathway for the biodegradation of indole in Aspergillus niger

Indole and its derivatives form a class of toxic recalcitrant environmental pollutants. The growth of Aspergillus niger was inhibited by very low concentrations (0.005 to 0.02%) of indole, even when 125- to 500-fold excess glucose was present in the medium. When 0.02% indole was added, the fungus showed a lag phase for about 30 h and the uptake of glucose was inhibited. Indole was metabolized by a new pathway via indoxyl (3-hydroxyindole), N-formylanthranilic acid, anthranilic acid,2,3-dihydroxybenzoic acid, and catechol, which was further degraded by ortho cleavage. The enzymes N-formylanthranilate deformylase, anthranilate hydroxylase, 2,3-dihydroxybenzoate decarboxylase, and catechol dioxygenase were induced by indole as early as after 5 h of growth, and their activities were demonstrated in a cell-free system.

Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90

Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion Of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.

p-Hydroxyphenylacetate-3-hydroxylase. A two-protein component enzyme

p-Hydroxyphenylacetate-3-hydroxylase, an inducible enzyme isolated from the soil bacterium Pseudomonas putida, catalyzes the conversion of p-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate. The enzyme requires two protein components: a flavoprotein and a colorless protein referred to as the coupling protein. The flavoprotein alone in the presence of p-hydroxyphenylacetate and substrate analogs catalyzes the wasteful oxidation of NADH with the stoichiometric generation of H2O2. A 1:1 complex of the flavoprotein and coupling protein is required for stoichiometric product formation. Such complex formation also eliminates the nonproductive NADH oxidase activity of the flavoprotein. A new assay measuring the product formation activity of the enzyme was developed using homoprotocatechuate-2,3-dioxygenase, as monitoring the oxidation of NADH was not sufficient to demonstrate enzyme activity. The coupling protein does not seem to have any redox center in it. Thus, this 2-component flavin hydroxylase resembles the other aromatic hydroxylases in that the only redox chromophore present is FAD.

Study on degradation of H-acid by Alcaligenes latus isolated from textile industrial effluent

This study presents 100% degradation of H-acid under optimized conditions using Alcaligenes latus, isolated from textile industrial effluent. Gene/s responsible for H-acid degradation was/were found to be present on plasmid DNA. Addition of bipyridyl to incubated medium resulted in accumulation of terminal aromatic compound, suggesting that catechol may be terminal aromatic compound in degradation pathway of H-acid by A. latus. SDS-PAGE of cell free extracts showed two prominent bands close to molecular weight of catechol 1,2-dioxygenase.

Estudo das alterações biológicas causadas pela aderência de cepas de Escherichia coli enteroagregativa (EAEC) com macrófagos humanos ativados da linhagem U-937

Escherichia coli enteroagregativa (EAEC) é um patógeno relacionado ao desenvolvimento de quadros de diarréia aguda ou persistente. A resposta inflamatória induzida por EAEC está relacionada à liberação de interleucina 8, que atua estimulando a transmigração de neutrófilos através do epitélio. Os macrófagos, de forma similar aos neutrófilos, são células fagocíticas que produzem espécies reativas de oxigênio (ERO), como o peróxido de hidrogênio (H2O2). Neste trabalho, avaliamos as consequências da interação de diferentes cepas clínicas com macrófagos humanos ativados da linhagem U-937. Todas as cepas testadas apresentaram filamentos nos testes de aderência aos macrófagos, diferentemente do que ocorre na interação com outras linhagens celulares como HEp-2, T84 e Caco-2. O ferro é um microelemento essencial para bactérias, sendo utilizado como cofator de enzimas e que também pode participar da geração de ERO através da reação de Fenton. Considerando-se a possibilidade de que o H2O2 produzido pelos macrófagos possa gerar radical hidroxil através da reação de Fenton, testes de aderência foram realizados com as amostras cultivadas na presença do captador de ferro 2,2-dipiridil. Tal fato não suprimiu a formação de filamentos, entretanto diminuiu a aderência das cepas EAEC 042 e 17-2. Com o objetivo de produzir uma resposta adaptativa ao H2O2, as culturas bacterianas foram pré-tratadas com uma dose sub-letal de H2O2 por 60 minutos antes de aderirem aos macrófagos. Nossos resultados mostraram que o pré-tratamento também não inibiu o aparecimento de filamentos em relação às culturas não tratadas. Além disto, foi observado que o pré-tratamento com o H2O2 reduziu a aderência das amostras de EAEC ao tapete celular. A filamentação é uma das respostas SOS, induzida pela presença de danos e/ou bloqueio na síntese da molécula de DNA. Com o objetivo de verificar se o H2O2 produzido pelos macrófagos estaria causando danos induzindo o sistema SOS e a filamentação bacteriana, foram realizados testes de viabilidade com mutantes derivados de E. coli K12 deficientes em enzimas do reparo por excisão de bases (BW535) e na resposta SOS (DM49). Nossos resultados mostram que os mutantes apresentaram os níveis de sobrevivência semelhantes ao observado para cepa selvagem isogênica (AB1157). Todos estes resultados em conjunto indicam que o H2O2 não é o indutor da filamentação nos testes de aderência. Macrófagos ativados apresentam ação microbicida através da ação da enzima indolamina dioxigenase (IDO), associada à redução do aminoácido L-triptofano. Desta forma, realizamos testes qualitativos de aderência de EAEC aos macrófagos suplementando o meio de interação com este aminoácido. Nossos resultados mostram que a adição de triptofano ao meio de interação reduz o número de filamentos por campo. Desta forma, aventamos a hipótese de que a depleção do triptofano seja responsável pela indução de resposta SOS, tendo como conseqüência a filamentação das bactérias.

Species Variation in the Mechanisms of Mesenchymal Stem Cell-Mediated Immunosuppression

Bone marrow-derived mesenchymal stem cells (MSCs) hold great promise for treating immune disorders because of their immunoregulatory capacity, but the mechanism remains controversial. As we show here, the mechanism of MSC-mediated immunosuppression varies

Biodegradation of dibenzofuran by Janibacter terrae strain XJ-1

The dibenzofuran (DF)-degrading bacterium, Janibacter terrae strain XJ-1, was isolated from sediment from East Lake in Wuhan, China. This strain grows aerobically on DF as the sole source of carbon and energy; it has a doubling time of 12 hours at 30 degrees C; and it almost completely degraded 100 mg/L-1 DF in 5 days, producing 2,2',3-trihydroxybiphenyl, salicylic acid, gentisic acid, and other metabolites. The dbdA (DF dioxygenase) gene cluster in the strain is almost identical to that on a large plasmid in Terrabacter sp. YK3. Unlike Janibacter sp. strain YY-1, XJ-1 accumulates gentisic acid rather than catechol as a final product of DF degradation.

肿瘤耐药与心肌肥厚两种生物医学体系的比较蛋白质组学研究

随着人类基因组计划的完成，蛋白质组学研究已成为生命科学乃至自然科学中最活跃的学科领域之一。蛋白质组学将对我们理解后基因组学时代的基因功能有极大的帮助。差异表达蛋白质组学通过对不同样品蛋白质表达水平的比较，已经被广泛地应用在疾病的研究领域。P糖蛋白已经被证实参与介导了在急性粒白血病中发生的多药耐药，然而越来越多的研究结果表明，在急性粒白血病中多药耐药的机制是多因素的。为了寻找白血病肿瘤细胞中与耐药相关的靶蛋白，我们采用蛋白质组学研究技术，对多药耐药细胞系K562／A02的蛋白质表达谱进行了分析。 本研究采用蛋白质组学研究技术，对多药耐药细胞系K562／A02和药物敏感细胞系K562／S的总蛋白质进行双向凝胶电泳（2 DE）的差异分析，结果发现在K562／A02细胞中有41个蛋白质点差异显著，并用基质辅助激光解离飞行时间质谱（MALDI-MS）成功鉴定了大多数的蛋自点。在被鉴定出的差异蛋白质中，有一些是首次发现与耐药相关，包括chloride intracellular charmel，chaperonin，proteindisulfide-isomerase ER6o precursor and constitutive heat shock protein 70等。所得结果不但能够使我们更加深入的理解肿瘤耐药的病理机制，更重要的是通过找到肿瘤细胞发生耐药的标志性蛋白，可以为开发肿瘤耐药预测方法和寻找抗耐药肿瘤靶标新药的线索，同时也为计算机虚拟筛选设计抗耐药肿瘤新药奠定基础。除了详细的研究了K562／A02细胞系差异蛋白质组，我们还采用蛋白质组学的方法，以乳鼠心肌细胞为实验模型，研究在ARs激动的不同清况下心肌细胞蛋白质表达谱的变化。心肌肥厚是心脏工作超负荷的一种代偿反应。目前，越来越多的证据表明ARs在心肌肥厚的发生中起着非常重要的作用，但人们对其机制了解尚不全面。通过检测心肌肥厚时全蛋白表达的变化，将有助于我们全面、深入了解ARs在心肌肥厚中的作用机制。通过严格的2D胶图象分析，蛋白取点后胶内水解，高灵敏度的质谱鉴定及数据库检索，我们成功地进行了乳鼠心肌细胞肥厚为模型的蛋白质组学的研究。本实验通过比较研究不同肾上腺素受体激动剂norePinephrine伽E），phenylephxine（PE）and isoprenaline（ISO）诱导组与未诱导对照组的蛋白质组学变化，分析确定蛋白丰度差异在2倍以上的蛋白质点，并通过MALDI-MS进行蛋白鉴定。20个具有显著差异的蛋白质被鉴定，包括caspase-11，vimeniin，4-hydroxyphenylpyruvic acid dioxygenase，Modl protein，Txndcs protein等，其中一些蛋白在此之前并没发现与心肌肥厚相关，对它们在心肌肥厚中的意义和机制我们将做进一步的探讨。本研究结果将为我们更好理解肾上腺素受体之间以及肾上腺素受体与心肌肥厚之间的关系提供有益的线索，并将为最终发现可应用于临床的新的诊断和治疗心血管疾病的标志性蛋白打下基础。

中国沙棘(Hippophae rhamnoides subsp. sinensis)不同种群对干旱胁迫的响应差异与干旱诱导蛋白的分析

沙棘广泛分布于亚欧大陆的温带地区和亚洲亚热带的高海拔地区。沙棘能适应多种生态环境，能耐受多种逆境(如干旱、低温、高温和盐害等)。在中国，沙棘常常被用作植被恢复中的先锋树种而大量栽培。本文以中国沙棘为试验材料，探索沙棘适应干旱机制，以及沙棘对干旱胁迫的适应机制是否存在种群间的差异，同时试图通过分析干旱胁迫下沙棘叶片蛋白质表达变化探索沙棘适应干旱胁迫的分子机理。 对三个分别来自低海拔湿润地区、低海拔干旱地区和高海拔湿润地区的中国沙棘种群进行干旱胁迫处理。干旱胁迫能提高根冠比，比叶面积，降低平均叶面积和总生物量，提高沙棘的抗氧化性酶活性、脯氨酸含量、脱落酸(ABA)含量、降低光合作用，提高长期用水效率。实验中的这两个低海拔种群比高海拔种群抵抗干旱的能力更强,不同的种群采用了不同的策略来耐受干旱胁迫和过氧化胁迫。 在2004 年度的实验中，干旱胁迫处理下，高海拔湿润种群(道孚种群)严重失水，生长也受到更大的抑制，非气孔因素在抑制光合作用方面占支配地位，抗坏血酸含量下降，ABA和脯氨酸含量增加幅度比九寨沟种群的要高，这可能是因为道孚种群严重失水而引起的；而低海拔湿润种群(九寨沟种群)的体内水分状况几乎不受干旱的影响，生长情况也较道孚种群要好。 在2005 年度的试验中，和高海拔湿润地区种群(道孚)相比较，低海拔干旱地区种群(定西)在叶片相对水含量、根冠比、抗氧化酶活性(过氧化氢酶、抗坏血酸过氧化物酶和谷胱甘肽过氧化物酶)、保护性物质(脯氨酸，脱落酸)含量等方面都要高，光能热耗散能力也更强，而且气体交换参数(气孔扩散阻力和胞间CO2浓度等)对干旱也更不敏感。 分析了干旱胁迫下沙棘叶片蛋白质表达的变化。共发现319 个蛋白质，有4 个蛋白在干旱胁迫下消失(Putative ABCtransporter ATP-binding protein 、Hypothetical proteinXP-515578，热激蛋白Hslu219 和一个没得到鉴定的蛋白)，4 个只在干旱胁迫下出现(没命名的蛋白质产物，对甲基苯-丙酮酸双加氧酶，NTrX 和一个没得到鉴定的蛋白)，46 个蛋白质的表达丰度变化显著，包括32 个干旱负调蛋白，14 个干旱正调蛋白(3 个Rubisco 的大亚基、J-type–co-chaperone Hsc20、putative protein DSM3645-2335、putative acyl-COA 脱氢酶、nesprin-2 和两个没有得到鉴定的蛋白质)。这些蛋白质参与了氮代谢调控、抗氧化行物质的合成、脂肪酸β－氧化、核骨架构造、[Fe-S]基团组装、物质跨膜运输、细胞分裂或作为分子伴侣和蛋白质酶起作用。putative ABC transporter ATP-binging protein、NtrX、nesprin-2 和Hslu 是本试验新发现的高等植物蛋白，我们主要从它们的保守结构域或在其他生物中的同源物来猜测它们的功能。实验结果为我们研究植物抗干旱机制提供了新线索和新视野。 Seabuckthorn (Hippophae rhamnoides L.) is widly distributed throughtout the temperatureresiogn of Europe and Asia and sub-tropical plateau zone of Asia. H. rhamnoides can adapatseveral different environments, and can tolerant several envioronmental stresses (e.g, lowtemperature, high temperature, drought and salty). It has been widely used in forest restoration asthe pioneer species in China. In present study, we applied H.rhamnoides subsp. Sinensis asexperimental materials to study its drought-tolerant mechanism, and expected to findpopulational difference in drought-tolerant mechanism that may exist among populations, and tryto get some insight in drought-tolerant mechanism of it at morecular level through analyzing thechange of leaf protein expression. Three populations from high altitude wet zone, low altitude wet zone and low altitude arid znoe,respectively, were applied in our experiment, and were subjected to drought. Drought increasedthe root/shoot ratio(RS), special leaf area, long-term water use efficinency, activity of antioxidantenzymes, proline content and abscisic acid (ABA) content, declined the net photosynthesis rate(A), average leaf area (ALA), total biomass (TB). Both two low altitude populations were moredrought-tolerant than the high altitude population, and different population applied differentstratedgies to tolerant oxidant stress and drought stress. The results of the exprement in 2004 showed that Daofu population was more drought-sensitivethan Jiuzhai population. Under drought conditions, leaf relative water content (RWC) greatlydecreased in Daofu population, but not in Jiuzhai population. The large loss of water in Daofupopulation resulted in a limitation on A mainly caused by non-stomatal factors, severer suppression in growth rate and a significant reduction in ascorbic acid (AsA) content, comparedwith Jiuzhai population. The greater increase in content of ABA and proline in Daofu populationmay be also induced by large loss in water, so that enable plants to cope with sever drought. In the exprement of 2005, drought significantly increased RS, activities of catalase (CAT),peroxidase (POD), glutathione peroxidase (GPX) and ascorbate peroxidase (APX), and alsosignificantly increased ABA and proline contents. On the other hand, compared with Daofupopulation, drought induced larger RS and activities of CAT, GPX and APX, and higher ABAcontent in Dingxi population, whereas gas exchange traits, e.g., stomatal limitation value (LS) andintercellular CO2 concentration (Ci), were less responsive to drought in Dingxi population thanthose in Daofu population. All these factors enable Dingxi population to tolerant drought betterthan Daofu population. The leaf protein profile of seabuchthorn subjected to drought was analyzed. Altogether 319proteins were detected in well-watered sample, four proteins disappeard by drought (putativeABCtransporter ATP-binding protein, hypothetical protein XP-515578, Hslu219and aunidentified protein), four only appeared under drought (a probable nitrogen regulation protein(NtrX), a 4-hydroxyphenylpyruvate dioxygenase , an unnamed protein product and an identified protein), 32 drought down-regulated proteins, and 14 drought up-regulated proteins (nine wereidentified: three large subunits of Rubisco, a hypothetical protein DSM3645-23351, a putativeacyl-COA dehydrogenase, a nesprin-2, a J-type-co-chaperone HSC20 and two unmatchedproteins). These proteins may involve in β-oxidation, cross-membrane transport, cell division,cytoskeleton stabilization, iron-sulfur cluster assembly, nitrogen metabolism regulation andantioxidant substance biosynthesis or function as molecular chaperone or protease. Four proteins(a putative ABC transporter ATP-binging protein, NtrX, nesprin-2, Hslu) were new found in highplants, and their functions were estimated from their conserved domain or their homologues inother organism. Our results provided new clue and new insight for us to study thedrought-tolerant mechanism in plants.

菌剂强化处理炼油废水及苯酚冲击下功能菌响应机制研究

近年来，我国炼油行业发展迅速，炼油能力全世界第二，炼油行业已成为污染大户。本研究针对炼油废水生物处理中存在的稳定达标难、抗冲击负荷能力差、建设投资与运行成本高等问题，就菌剂强化处理炼油废水中试与工程应用展开了研究，以期为菌剂的工程应用与推广提供理论参考与技术支持；并以炼油废水中的主要特征污染物苯酚为研究对象，考察了不同浓度苯酚冲击下功能菌的响应机制，并以此为指导研制功能菌激活促进剂，考察其对功能菌生物学指标的调控效果，以期为废水生物处理有毒污染物冲击调控提供理论依据与技术支持。 中试研究表明，菌剂强化处理炼油废水，出水COD、NH4+-N 平均值为86.7、7.6 mg/L，其平均去除率较常规生物处理系统分别提高了35.47%、59.28%，其耐受COD、NH4+-N 容积负荷分别高达2.42、0.139kg/(m3·d)，具有良好的耐冲击能力。工程应用研究表明，菌剂强化处理炼油废水，出水COD、NH4+-N 平均值分别为85.05、8.4mg/L，其去除率较常规生物处理系统提高了25.1%、28.7%，出水水质各项指标均达到了国家《污水综合排放标准GB 8978-1996》一级排放标准。技术经济分析表明，菌剂强化处理炼油废水在建设成本、运行成本上分别降低38%、49%，具有良好的技术经济优势。 苯酚冲击下功能菌响应机制研究表明：不同浓度苯酚冲击下，生物学指标生物量、脱氢酶酶活、1,2-双加氧酶酶活对冲击都有不同程度的响应，其响应敏感程度为脱氢酶酶活＞生物量＞1,2-双加氧酶酶活。1,2-双加氧酶酶活与COD 降解率相关性良好，可表征苯酚降解过程，确认为调控重点。以此为指导研制出苯酚降解功能菌抗冲击激活促进剂，可有效调控功能菌对有毒污染物苯酚的降解效果，1000mg/L 苯酚冲击下，经调控，其COD 去除率较对照提高20%，降解时间缩短16%以上。其对生物学指标的调控效果为1,2-双加氧酶酶活＞生物量＞脱氢酶酶活，验证了功能菌在苯酚冲击下的响应机制。研究表明菌剂强化处理炼油废水切实可行，具有良好的技术经济优势。有毒污染物冲击下废水生物处理系统响应机制研究为抗冲击调控提供了新的研究思路。 Currently, China’s oil refining industry is developing rapidly and has become the second largest all over the world. The oil refining industry is one of the major pollution industries in our country. The pilot scale study and engineering application research were conducted aiming at the problems in refining wastewater such as poor treatment stability and water quality, poor anti-shock capacity and expensive running cost, etc., so as to provide theoretical references and technological supports for the engineering application and popularization of microbial preparation in wastewater treatment. Also, the response mechanism of functional microbe under shock of different phenol concentrations, which is the main pollutants in refinery wastewater, was studied. Based on this result, functional microbe activation accelerator was developed, and the regulation effect of functional microbe biological index under phenol shocking were studied, in order to provide theoretical basis and technological support for regulation of toxic shocking of wastewater biological treatment. The result of pilot scale research indicated: for treatment of refinery wastewater in bioaugmention treatment system of microbial preparation, the COD and NH4+-N average value of effluent was 86.7 and 7.6 mg/L, Comparing with normal biological treatment system, the average removal rates of COD, NH4+-N increased 35.47%,59.28% separately by bioaugmention treatment system, which showed better anti-shocking capacity, the volumetric load r of COD and NH4+-N reached 2.42 kg/(m3·d) and 0.139 kg/(m3·d), respectively. The research on engineering application of refinery wastewater bioaugmentation treatment by microbial preparation indicated:the average concentrations of effluent COD and NH4+-N in the bioaugmentation treatment system were 85.05 and 8.4mg/L, which increased by 25.1% and 28.7% comparing with normal biological treatment system of refinery wastewater, And the effluent quality meets the first grade of discharging standard of National Integrated Wastewater Discharge Standard GB 8978-1996. The economic analysis of technology indicated: the demonstration project of bioaugmentation treatment of refinery wastewater by microbial preparation decreased by 38% in construction cost and 49% in running cost. This technology has economic benefits. The response mechanism of functional microbe under phenol shock indicated: biological index such as the biomass concentration, dehydrogenase and 1,2-dioxygenase had different responses under phenol shocking of different concentrations. The response sensitivity of different biological index under phenol shocking of different concentrations is: dehydogenase activity ＞ biomass ＞1,2-dioxygenase activity, and high correlation of 1,2-dioxygenase and COD degradation percentage is achieved, thus 1,2-dioxygenase could be used to reflect the degradation situation of pollutants. So, 1,2-dioxygenase is the keypoint of regulation. The anti-shock activation accelerator of phenol degradation functional microbe was primarily developed. The results indicated: the activation accelerator could regulate the degradation effect of toxic substance-phenol by functional microbe effectively. For the functional microbe treatment system under phenol shocking of 1000mg/L, the COD degradation rate increased by 20% and the degradation time reduced by more than 16% under regulation of activation accelerator. The regulation effects of biological index are: 1,2-dioxygenase ＞ biomass ＞ dehydrogenase. In this way, the response mechanism of functional microbe under toxic shocking is verified. The result indicated: the augmented microbial preparation treatment of refinery wastewater is applicable. It has many technical and economical advantages. The research results of responses mechanism of wastewater treatment system on toxic pollutants would offer a new idea for regulation of anti-shock.