Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species-Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast.

Estudo aberto prospectivo de 30 pacientes com onicomicose por Scytalidium spp. tratados com terbinafina oral, ciclopiroxolamina esmalte e onicoabrasão

Onicomicose é um termo geral usado para definir infecção fúngica da unha. Seus agentes podem ser dermatófitos, leveduras ou fungos filamentosos não dermatófitos - FFNDs. Estes são comumente encontrados na natureza como saprófitas do solo e de restos vegetais e patógenos de plantas e têm sido considerados fungos patógenos primários de lesões cutâneas. Não existe até o momento terapêutica padrão para o tratamento de onicomicoses por Scytalidium spp., sendo escassos os dados na literatura pesquisada. Este trabalho tem como objetivo avaliar e comparar a resposta terapêutica a três abordagens diferentes de tratamento combinado para onicomicose por Scytalidium spp., todos associados à onicoabrasão. Foram selecionados 30 pacientes com diagnóstico de onicomicose provocada por Scytalidium spp., divididos em três grupos de dez, recebendo cada um os seguintes tratamentos, além da onicoabrasão: Grupo I: Terbinafina oral e esmalte de ciclopiroxolamina 8%, duas vezes por semana por 12 meses; Grupos II e III: Esmalte de ciclopiroxolamina 8%, duas e 5 vezes por semana, respectivamente, por 12 meses. Os parâmetros de avaliação da eficácia foram clínico e micológico ao término do tratamento e seis meses após. Foram utilizados os critérios de cura total, cura parcial, falha terapêutica aos 12 meses e recidiva/reinfecção no acompanhamento de seis meses. Vinte e cinto pacientes completaram o estudo. Não houve diferença estatística entre os grupos nos diversos parâmetros utilizados para avaliação da resposta terapêutica. A avaliação do resultado terapêutico mostra que ao final de 12 meses de tratamento apenas um paciente preencheu os critérios para cura total, e que 32% dos pacientes de todos os grupos apresentaram cura parcial. Todos os pacientes que completaram o estudo obtiveram melhora clínica, que se manteve no período de acompanhamento. A presença dos fungos na lâmina ungueal foi constante, mesmo com a melhora clínica. Embora não se possa afirmar qual a melhor forma de intervenção entre as três terapêuticas propostas devido ao pequeno número de pacientes do estudo, deduz-se, deste trabalho, que não houve vantagem na administração de terapia sistêmica concomitante. É possível considerar que a terapia tópica exclusiva, seja duas ou cinco vezes por semana, possa constituir opção mais adequada para o tratamento da onicomicose por Scytalidium spp.

Fermentação alcoólica de hidrolisado de farelo de mandioca usando levedura Saccharomyces cerevisiae álcool resistente

A matriz energética mundial é baseada em fontes fósseis e renováveis. No Brasil, o bioetanol é gerado principalmente a partir da cana-de-açúcar. Resíduos agroindustriais (fontes celulósicas ou amiláceas) despontam como biomassas alternativas à cana-de-açúcar, para aumentar a competitividade deste combustível renovável frente aos de origem fóssil e também favorecer a sustentabilidade e a segurança alimentar e energética, pois são ricos em polissacarídeos não diretamente fermentescíveis, abundantes (problema ambiental) e apresentam baixo valor comercial. O farelo de mandioca é um exemplo de resíduo sólido gerado na produção de fécula (amido) e farinha de mandioca que ainda contém, em média, 75% de amido. Consequentemente, deve ser previamente hidrolisado e posteriormente fermentado por leveduras do gênero Saccharomyces para gerar etanol. O objetivo deste estudo foi produzir bioetanol a partir de hidrolisados enzimáticos de farelo de mandioca, usando levedura álcool resistente (AR). Primeiramente, a concentração de açúcares obtida a partir da hidrólise enzimática foi verificada através de um planejamento fatorial completo (24), com triplicata no ponto central, a fim de investigar a influência dos seguintes fatores na hidrólise: concentração de α-amilase (Termamyl 2X), tempo de liquefação, concentração de glucoamilase (AMG 300L) e o tempo sacarificação. A condição de hidrólise mais favorável foi a do ensaio com 0,517 mL de AMG/g amido, 0,270 mL de Termamyl/g amido, 1h de tempo de liquefação e 2h de tempo de sacarificação. O caldo resultante da condição escolhida alcançou altas concentrações de glicose (160 g/L). Os ensaios de fermentação alcoólica foram realizados em duplicata em biorreator de 3L, em regime de batelada, a 30C, 100 rpm e pH 5,5. Cerca de 3 g/L (massa seca) de uma linhagem de levedura álcool tolerante, Saccharomyces cerevisiae Hansen BY4741, crescida por 12h em meio YEDP (2% de glicose) foram usados como inóculo. O mosto consistiu de um litro de hidrolisado (160 g/L de glicose) fortificado com extrato de levedura (1%) e peptona de carne (1%), além da adição de um antiespumante (Tween 80) na concentração de 0,05% (m/v). Em 30 horas de fermentação, a média da concentração de etanol obtida foi de 65 g/L. A eficiência foi de 87,6% e o rendimento e a produtividade foram 0,448 e 2,16 g/L.h, respectivamente. Os resultados indicaram a aplicabilidade do farelo de mandioca como matéria-prima para a produção de bioetanol

提高生物拮抗菌对果实采后病害的生防效力研究

摘要 　　"随着人们对身体健康和环境污染的日益重视，化学农药作为控制果实采后病害的主要方法受到了很大限制，科学研究者不得不寻求更加安全有效的防治果实采后病害的新方法。生物防治以其对环境和人类健康不造成危害的优点而逐渐受到人们的青睐。然而，由于生物防治是以活菌为基础，有其局限性和时效性，单独使用拮抗菌很难达到化学药剂完全控制果实采后病害的效果，因此，提高拮抗菌的生防效力成为当今生物防治领域的研究重点。本文主要研究了拮抗菌与不同外源物质配合使用的抑病效果及协同抑病机理;拮抗菌对采前田间和采后贮藏环境条件的适应能力;以及采前应用拮抗菌对果实采后贮藏期间病害的生物防治效力。研究结果表明： 　　1、酵母拮抗菌Cryptococcus laurentii与低浓度化学杀菌剂imazalil（25g/ml）和kresoxim-methyl（50g/ml）配合使用可以显著提高对冬枣果实采后黑霉病（Alternaria alternata）和褐腐病（Monilinia fructicola）的防治效果，杀菌剂并不影响拮抗菌在冬枣果实伤口的生长动态。 　　2、酵母拮抗菌Pichia membranefaciens和C. laurentii 与钼酸铵（NH4-Mo，5 mmol/L）和碳酸氢钠（NaHCO3，2%）配合能够显著提高对甜樱桃果实采后褐腐病（M. fructicola）的抑病能力。通过in vitro和扫描电镜观察结果表明，NH4-Mo和NaHCO3能够显著地抑制病原菌M. fructicola在培养基和果实伤口的生长，具有杀菌作用。 　　3、酵母拮抗菌C. laurentii和Rhodotorula glutinis与硅酸钠（Na2SiO3）配合使用对甜樱桃果实采后青霉病（Penicillium expansum）和褐腐病（M. fructicola）以及对冬枣果实青霉病（P. expansum）和黑霉病（A. alternata）的防治效果更好。经in vitro和扫描电镜观察表明，Na2SiO3对病原菌在培养基和果实伤口的生长有明显的抑制作用。同时，Na2SiO3还能诱导果实苯丙氨酸解氨酶（PAL）、多酚氧化酶（PPO）和过氧化物酶（POD）等抗性相关酶活性的提高。 　　4、酵母拮抗菌R. glutinis与水杨酸（SA，0.5mmol/L）配合可显著提高对甜樱桃果实采后青霉病（P. expansum）和黑霉病（A. alternata）的抑病能力。SA不影响拮抗菌在果实伤口的生长，in vitro实验中低浓度的SA对病原菌孢子萌发和芽管伸长也没有抑制作用。SA可能是通过诱导果实产生抗性来协同提高拮抗菌的抑病效果，而不是直接抑制病原菌生长。 　　5、酵母拮抗菌C. laurentii和R. glutinis在气调（Controlled atmospheres, CA）贮藏条件下对樱桃果实采后青霉病（P. expansum）和黑霉病（A. alternata）的防治效果显著提高。气调贮藏不抑制拮抗菌在甜樱桃果实伤口的生长。 　　6、采前应用酵母拮抗菌C. laurentii 和R. glutinis能够显著抑制甜樱桃果实在采后不同贮藏环境下的发病率。拮抗菌能够在田间果实表面生长并一直保持较高的数量。在试验的三种酵母拮抗菌中，C. laurentii的防病效果最好，该菌不仅能在果实表面迅速生长，也能适应低温和CA贮藏环境。"

酵母拮抗菌的抑病机理及生防效力改良的研究

相对于酵母拮抗菌的使用来说，人们对其作用机理了解得还不是很清楚。而了解拮抗菌的抑菌机理却是增强拮抗菌的生防效果以及进行拮抗菌筛选标准的重要前提。本文主要研究了酵母拮抗菌Pichia membranefaciens、Cryptococcus albidus以及Crytococcus laurentii对水果采后软腐病、褐腐病以及青霉病的防治效果，拮抗菌与病原菌之间的相互作用，并对酵母拮抗菌与外源物质配合使用，以及通过遗传改良途径来提高酵母拮抗菌生防能力等进行了初步研究。实验结果如下： 1、酵母拮抗菌P. membranefaciens、C. albidus以及C. laurentii能在果实伤口大量繁殖。采用扫描电镜技术，观察发现在桃果实伤口处P. membranefaciens能紧密地吸附在软腐病菌Rhizopous stolonfier的菌丝体上;C. laurentii与青霉病菌Penicillium expansum在苹果果实伤口处也存在着直接的拮抗作用;但P. membranefaciens和C. albidus对P. expansum的直接作用不明显。 2、酵母拮抗菌P. membranefaciens能够有效地抑制甜樱桃果实在常温和低温贮藏条件下褐腐病的发生。在常温贮藏条件下，P. membranefaciens和褐腐病菌Monilinia fracticola 处理都能够提高果实β-1,3-葡聚糖酶、POD、以及PAL酶的活性，但在低温贮藏条件下，拮抗菌和病原菌处理对甜樱桃果实β-1,3-葡聚糖酶、POD酶活性的升高有促进作用，对PAL和PPO酶活性的诱导作用不明显。 3、梨果实采后经过水杨酸，CaCl2，UV辐射和草酸等各种激发子处理以后，再接种病原菌Alternaria alternata，可以显著降低梨果实的发病率。其中，水杨酸处理的果实发病率最低。不同的激发子均可以诱导梨果实β-1,3-葡聚糖酶、POD、PAL和PPO酶活性的升高，但对果实乙烯含量的影响不明显。 4、氨基糖甙类抗菌素G418能够抑制P. membranefaciens的生长，其最低抑制浓度为100g ml-1。将G418抗性基因Neor插入到酵母-大肠杆菌穿梭表达载体pFL61中，构建PGK启动子驱动的表达载体pFL61-neo，利用醋酸锂转化法转化P. membranefaciens。酵母转化子在非选择性培养条件下连续生长50代后，仍有67.87%的细胞保留该质粒。这表明穿梭表达载体pFL61-neo能稳定地存在于P. membranefaciens中，并且该酵母细胞能有效地识别PGK启动子和终止子指导Neor的表达。 5、酵母拮抗菌C. laurentii和Rhodotorula glutinis与2%的碳酸氢钠混合使用，对冬枣果实青霉病的防治效果明显比单独使用拮抗菌或化学物质的防病效果好。其中，107CFU ml-1的拮抗菌与238 mmol l-1的碳酸氢钠配合使用可以达到单独使用108CFU ml-1拮抗菌的防病效果。另外，钼酸铵作为一种添加剂也能提高R. glutinis对梨果实青霉病和黑霉病的防治效果，但将钼酸铵与Trichosporon sp.配合使用的防病效果不明显。碳酸氢钠和钼酸铵在果实伤口对酵母拮抗菌的生长都有一定的抑制作用。 6、酵母拮抗菌P. membranefaciens在不同碳源、氮源中生长情况表明：在几种氮源中，大豆蛋白胨、酵母提取物、牛肉浸膏对P. membranefaciens的生长有显著的促进作用，其中，大豆蛋白胨的效果最好。在检测以葡萄糖、果糖和麦芽糖作为碳源的生长实验中，发现这几种碳源都能够被拮抗菌很好的利用，其中葡萄糖的利用率最好。小球藻生长因子（CGF）能够明显地促进了P. membranefaciens的生长。但是，CGF的浓度从0.5%增加到1%并没有促进酵母菌细胞数量的增加。

酵母拮抗菌的抑病效力、规模化培养及相关机理研究

我们实验室从果实表面分离获得的酵母拮抗菌已经证明能有效防治各种果实采后主要病害，为了加快生物拮抗菌的商业化应用，本文在完善拮抗菌抑病机理的基础上，重点研究了拮抗菌规模化培养条件，生物菌剂制品的稳定性，以及拮抗菌对环境胁迫的生理反应。主要研究内容包括：（1）分析酵母菌拮抗菌、病原菌与果实之间的互作效应及其影响因子;（2）筛选酵母拮抗菌规模化培养的最佳营养配方及培养条件;（3）优化酵母拮抗菌干粉与液体剂型的制备方式;（4）研究酵母拮抗菌在不同剂型中生活力下降的可能机理;（5）探讨酵母拮抗菌次生代谢产物的抑菌效果。研究结果如下： 1、单独接种Monilinia fructicola或同时接种M. fructicola和Cryptococcus laurentii均能诱导甜樱桃果实SOD、CAT和POD等抗氧化酶活性升高并加速脂质过氧化，同时伴有PPO同工酶新酶带出现。病原菌M. fructicola和Penicillum expansum在接种初期均显著促进拮抗菌C. laurentii在桃果实伤口处的生长。C. laurentii在接种24h内显著抑制桃果实LOX活性、O2•-产生与H2O2积累。单独接种病原菌能显著诱导桃果实LOX活性升高，促进O2•-产生，但抑制H2O2积累。病菌侵染后果实中 O2•-增加，以及H2O2的降低可能是桃果实对病原菌侵染的一种生理应答方式。 2、抗坏血酸钠能显著提高C. laurentii对甜樱桃果实褐腐病的防治效果，较低浓度的拮抗菌（ 1×107 cells mL-1）与200mM抗坏血酸配合使用可以达到较高浓度拮抗菌（1×108 cells mL-1）单独使用对M. fructicola的防治效果。抗坏血酸钠的协同抑病机理可能是在抑制病原菌生长的同时，也抑制了果实的抗氧化酶活性，从而加速了脂质过氧化过程。 3、酵母菌产业化培养条件的筛选结果表明，不同拮抗菌对培养基中营养物质的需求不一样，培养所需的温度有差异。在120L发酵罐的中试实验表明两种拮抗菌采用筛选出的最佳培养条件均得到浓度大于1× 109 CFU mL-1的菌悬液。 4、保护剂种类是影响Rhodotorula glutinis 和 C. laurentii两种酵母拮抗菌冷冻干燥效果的最主要因素，但保护剂效果的发挥依赖于其浓度与酵母菌生长阶段。无菌水和PBS（100mM, pH5.8）可以作为拮抗菌C. laurentii液体剂型的有效保护剂，而柠檬酸钠（100mM, pH5.8）则诱导拮抗菌C. laurentii的生活力快速丧失。 5、酵母菌拮抗菌冻干制品的研究表明，在胁迫环境下酵母菌生活力快速丧失与大量产生活性氧有关，这暗示活性氧的产生可能是导致酵母菌细胞死亡的主要因素。柠檬酸钠（100mM, pH5.8）对C. laurntii死亡的诱导效应受柠檬酸根浓度和介质酸度的双重影响。活性氧在柠檬酸钠诱导酵母菌生活力快速丧失中大量产生并发挥重要作用。 6、酵母拮抗菌能够产生某些对果实采后病原真菌具有抑制效果的挥发性和不挥发性物质。同一种酵母菌产生的物质对不同病原菌有不同的拮抗效果，而不同酵母菌对同一种病原菌的拮抗效果也不完全相同。但是，不同类型的培养基对拮抗菌产生的抑菌物质有明显的影响。

果实对酵母拮抗菌和外源水杨酸诱导的抗病性应答机理

用生物和非生物因子来进行采后病害的防治，是一个非常有效的方法。诱导抗性作为控制果蔬采后病害的生物技术，已成为该领域的一个研究热点。然而诱导抗性的机制非常复杂，涉及到寄主、病原菌、激发子之间的相互作用关系。本研究主要利用酵母拮抗菌Pichia membranefaciens和SA处理果实，观察其抗性诱导表达和对采后青霉病菌（Penicillium expansum）的抑制作用，并从蛋白质组学水平上对诱导抗性的机理进行了分析。研究结果表明： 1、酵母拮抗菌P. membranefaciens (5 × 107 cells·ml-1)和SA(0.5 mM)处理采后甜樱桃果实，能够明显地降低病害的发病率和病斑直径。酵母菌和SA处理影响到了果实抗氧化酶的活性，同时还改变了POD同工酶谱和甜樱桃果实的总蛋白含量，并诱导了新的蛋白质条带产生。用光学显微镜和扫描电子显微镜技术观察发现，在in vitro条件下P. membranefaciens能够紧密地结合与病原菌的菌丝，而在in vivo条件下这种结合较为松散。 2、借鉴其它模式植物的方法，我们建立了一整套适用于多汁类植物材料的蛋白质组学研究方法。对于芒果，桃，甜樱桃、苹果以及冬枣等果实，都取得了重复性非常好的2-D图谱。我们应用该技术进一步研究了P. membranefaciens (1 × 108 cells·ml-1)以及SA (0.5 mM)处理对桃果实蛋白质组的诱导影响。结果显示，两种激发子处理都能够诱导桃果实产生抗性，从而减轻青霉病引起的腐烂。在诱导处理1 d以后，酵母拮抗菌和SA分别诱导22和16个蛋白的差异表达。质谱鉴定的蛋白属于6大类：代谢，防御反应，转录，能量途径以及细胞结构。有6个蛋白受到两种激发子的共同调控。其中，4种蛋白(包括glutathione peroxidase, polyphenol oxidase precursor, catalase和methionine sulfoxide reductase) 属于抗氧化蛋白，涉及到活性氧代谢。另2个蛋白（Major allergen Pru av 1和peroxidase）是病程相关蛋白，直接参与植物的防御反应。同时一些磷酸化酶和转录因子也受到两种激发子的调节从而参与果实的抗病反应。酶学测定和Northern杂交的结果表明，拮抗菌与SA处理均能影响过氧化氢酶活性及其基因的表达。 3、采前用较高浓度SA (2 mM) 短时间（10s）处理不同成熟期的甜樱桃果实，能够明显降低果实青霉病的病斑直径，并能减轻较低成熟度果实的发病率。在没有接菌的情况下，SA诱导了33个差异表达的蛋白，其中用质谱鉴定出了26个。而在接种病原菌的情况下，SA诱导了19个差异表达的蛋白，并鉴定出了其中的12个。这些蛋白分别涉及到代谢、防御反应、转录、能量途径、信号转导等过程。在没有接种病原菌的情况下，SA处理诱导了Putative DnaJ heat shock protein, PR1-like protein, Peroxidase, Major allergen Pru av 1 (Pru a 1)和Catalase等与抗病有关的蛋白。而在接种病原菌的情况下，诱导了PR1-like protein, Peroxidase和Catalase蛋白的差异表达。通过酶活性测定以及对细胞学定位的研究，我们发现在没有接种病原菌的情况下，POD的活性受到SA的诱导。但是在接种病原菌以后，诱导效果不明显。

海藻糖提高酵母拮抗菌生活力和生防效力的作用机制

近年来，利用酵母拮抗菌进行果实采后病害的生物防治已经成为果实采后领域的研究热点。但是，在实际应用中生物拮抗菌制剂的防病效果远不如化学药剂稳定。由于生物拮抗菌是活体，生活力和生防效力易受诸多因素影响。在商品化制剂的剂型加工、销售、以及使用过程中的环境条件往往影响酵母拮抗菌的生活力和抑病能力，成为酵母拮抗菌产业化生产和商品化应用过程中的一个主要障碍。将酵母拮抗菌和其它化学物质配合使用可以提高拮抗菌的生防效力。另外，增加酵母拮抗菌对逆境条件的耐受力也是增加或稳定其防治效果的有效途径。本文研究了海藻糖与酵母拮抗菌生活力和生防效力的关系，通过生理手段提高了酵母拮抗菌内源海藻糖的含量，同时探讨了在多种逆境条件下内源海藻糖含量对酵母拮抗菌生活力和生防效力的影响及其作用机制。主要研究结果如下： 1. 以1 %海藻糖作为碳源培养酵母拮抗菌Cryptococcus laurentii可以提高其内源海藻糖含量。在in vitro试验中，提高内源海藻糖含量可以提高C. laurentii在低温（1 ºC）、气调（1 ºC，5 % O2，5 % CO2）条件下的生活力;在in vivo试验中，提高C. laurentii内源海藻糖含量可以提高其在苹果果实伤口上的种群密度和对苹果青霉病的防治效果。内源海藻糖的积累还能提高C. laurentii冷冻干燥后的生活力，海藻糖对酵母细胞质膜的保护作用可能是一个主要原因。 2. 以1 %海藻糖作为碳源能提高酵母拮抗菌Rhodotorula glutinis的内源海藻糖含量。内源海藻糖含量的增加可以提高C. laurentii和R. glutinis在慢速冷冻处理中的生活力。同时，海藻糖作为外源保护剂可以明显提高两种酵母拮抗菌在冷冻干燥处理后的生活力。在快速冷冻、慢速冷冻和冷冻干燥处理中，提高酵母拮抗菌的内源海藻糖含量并使用海藻糖作为外源保护剂可以获得更高的生活力。同时，这种内、外源保护因子的综合作用也可以提高两种拮抗菌在苹果果实伤口上的种群密度和对苹果青霉病的防治效果。 3. 脱脂牛奶和糖（葡萄糖，半乳糖，蔗糖，海藻糖）作为保护剂可以提高C. laurentii冷冻干燥后在常温（25 ºC）和低温（4 ºC）保存过程中的生活力。脱脂牛奶和糖保护剂的复合使用对C. laurentii的保护效果高于其单独使用的效果。通过对几种常用的碳源进行筛选，发现柠檬酸作为碳源对C. laurentii内源海藻糖的积累有明显的诱导作用。当使用相同的保护剂或保护剂组合时，高内源海藻糖含量的酵母拮抗菌生活力更强。当冷冻干燥前使用半乳糖 + 脱脂牛奶作为保护剂时，高内源海藻糖含量的C. laurentii在4 ºC保存90 天后对苹果青霉病的防治效果和与新鲜培养的酵母拮抗菌相当。 4. 酵母拮抗菌C. laurentii冷冻干燥后在常温(25 ºC)保存期间，细胞生活力下降，细胞膜的完整性降低，胞内活性氧水平增加，同时与抗氧化相关的超氧化物歧化酶（SOD）活性也增加，而过氧化氢酶（CAT）活性则下降。提高内源海藻糖含量和/或使用外源保护剂（5 %脱脂牛奶 + 10 %葡萄糖）可以减缓上述指标下降或上升的速度。内、外源因子的共同作用有利于提高对拮抗菌细胞的保护作用。 5. 利用褐藻酸钠制成的含酵母拮抗菌的胶球在干燥后能有效的保持C. laurentii的生活力。在3种不同粘度的褐藻酸钠中，0.5 % 3500 cp的褐藻酸钠表现出较好的保护效果。内源海藻糖的积累可以提高干胶球中C. laurentii的生活力，作为外源保护剂的4种糖（葡萄糖、半乳糖、蔗糖、海藻糖）中只有海藻糖在低温条件下可以提高C. laurentii的生活力，其它3种糖反而降低了C. laurentii的生活力。

外源物质对酵母拮抗菌生活力的影响

　　近年来，酵母拮抗菌在水果采后病害防治中展示了良好的应用前景。然而，在实际应用中,酵母拮抗菌在逆境条件下会因为发生凋亡或细胞损伤而引起生活力的下降，最终导致拮抗菌抑病能力降低。研究酵母拮抗菌生活力下降的规律，提高酵母拮抗菌的生产效率，减少剂型加工过程中的细胞损伤，增强其对逆境条件的耐受力是增加或稳定生防制剂防治效果的有效途径。本文主要研究酵母拮抗菌正常培养过程中生活力下降的规律，筛选剂型加工过程中对酵母拮抗菌具有保护作用的化学物质，并对酵母拮抗菌的培养条件进行了优化。主要研究结果如下： 　　1. 在正常培养过程中，酵母拮抗菌Rhodotorula glutinis和Cryptococcus laurentii中细胞染色质凝集或细胞膜破损的发生一般在6天以后。外源加入的N-乙酰半胱氨酸及硅酸钠等物质在超过一定浓度时会加速酵母菌的死亡。 　　2. 在不同的液体悬浮制剂中，对R. glutinis而言，使用磷酸缓冲液（PBS）悬浮时保护效果最好；而C. laurentii悬浮在NYDB培养基中或海藻糖、乳糖溶液中时的生活力最高。 　　3. 以10 %葡萄糖 + 5 %脱脂牛奶作保护剂，可以有效地保持酵母拮抗菌C. laurentii冻干制剂的生活力，配合使用的保护效果高于它们单独使用时的保护效果。添加1 mM N-乙酰半胱氨酸能更好地保持拮抗菌制剂在常温保存过程中的生活力，这可能与这种还原性物质缓解了细胞内活性氧的积累有关。 　　4. 不同酵母拮抗菌对不同碳、氮源的利用能力有明显差异。在9种不同的碳源和10种不同的氮源中，Pichia membranefaciens能够最有效利用的碳、氮源是葡萄糖、果糖和多价胨，而Candida guilliermondii的最佳碳源和氮源分别是果糖和肉蛋白胨。

酵母拮抗菌和外源水杨酸对核果类果实抗病相关基因的诱导表达

在果实采后贮藏过程中，病原真菌的侵染会引起果实腐烂，造成巨大的经济损失。利用生物和非生物因子诱导果实抗病性，已经成为采后病害防治领域的一个研究热点。本文主要利用RT-PCR和RACE技术克隆果实抗病相关基因，通过分子杂交和蛋白羰基化免疫检测技术，研究了外源SA和酵母拮抗菌诱导果实抗病性机理，结果表明： 1. 通过优化RNA提取方法，能从含有多糖的冬枣、葡萄、甜樱桃、桃、番茄等果实中提取到质量较好的RNA，用于RT-PCR和Northern杂交。 2. 采用RT-PCR和RACE方法，从甜樱桃果实克隆了两个抗氧化相关基因CAT2（Genbank：EF165590）和GPX（Genbank：EF165591）和两个PR基因GLU-1（Genbank：EF177487）和GLU-3（Genbank：EF177488)。其中CAT2全长cDNA序列为1479 bp，编码492个氨基酸；GPX全长cDNA序列为513 bp，编码170个氨基酸；GLU-1全长cDNA序列为1050 bp，编码349个氨基酸；GLU-3部分cDNA序列为454 bp，编码141个氨基酸。 3. 酵母拮抗菌Pichia membranaefaciens处理不同成熟度的甜樱桃果实，能显著降低果实贮藏期间青霉病（Penicillium expansum）的发生，并且对低成熟度果实的病害防治效果更为明显。酵母拮抗菌的抑病机理与减轻了甜樱桃果实蛋白羰基化程度，诱导了果实抗氧化酶基因（CAT和GPX）和PR基因（GLU-1）的表达和提高了抗氧化酶（CAT和GPX）和β-1,3-葡聚糖酶的活性有关。 4. 四种酵母拮抗菌P. membranaefaciens, Cryptococcus laurentii, Candida guilliermondii和Rhodotorula glutinis处理桃果实，可显著降低贮藏期间的褐腐病（Monilinia fructicola）。这是由于酵母拮抗菌能抑制病原菌侵染造成的氧化胁迫和蛋白羰基化。此外，酵母拮抗菌处理还能显著诱导CAT、POD、几丁质酶、β-1,3-葡聚糖酶活性及相应基因的表达。 5. 水杨酸（SA，2 mM）处理采后不同成熟度的甜樱桃果实，能显著降低青霉病的危害。其抑病机理与SA处理能减轻P. expansum侵染引起的果实蛋白羰基化程度，显著提高CAT、GPX和β-1,3-葡聚糖酶基因的表达和相关的酶的活性有关。而2 mM的SA处理对P. expansum的生长没有直接抑制作用。 6. 水杨酸（SA，2 mM）与P. membranaefaciens（1×108 CFU/ml）配合处理能显著降低低温贮藏期间桃果实的褐腐病，并能提高几丁质酶、β-1,3-葡聚糖酶和POD的活性和相关基因的表达。另外，2 mM的SA对拮抗菌P. membranaefaciens的生长没有影响，但能够抑制病原菌M. fructicola的孢子萌发和菌丝扩展。

Bacterial flora of fresh and iced Indian mackerel (Rastrelliger kanagurta) and its response to chlortetracycline (CTC) treatment

Quantitative and qualitative studies on the bacterial flora of fresh Indian mackerel (Rastrelliger kanagurta) have been made. The total native flora as well as 5 ppm CTC insensitive flora of the fish showed variations with season. About 90% of the fresh fish flora was sensitive to 5 ppm CTC. The natural flora of the fresh fish consisted of Vibrios, Pseudomonas, Achromobacter, Flavobacterium, Corynebacteria, Micrococci, Bacillus and yeasts. In the CTC insensitive flora, Vibrios predominated followed by yeasts. The selection of bacterial genera during storage of the fish in ice and in 5 ppm CTC incorporated ice has also been investigated. At the time of spoilage, Pseudomonas was found to be the dominant flora of the fish stored in both types of ice.

无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因表达载体的构建及其在毕赤酵母中的表达

研究背景与目的：近二十年来，抗生素的广泛使用以及一些不当应用导致临床上出现大量的耐药性病原菌，所以不易产生耐药性的抗菌肽就成为目前研究的热点。本课题组此前的研究表明无指盘臭蛙（Odorrana grahami）皮肤抗菌肽具有广谱抗菌活性，但对真核细胞没有毒性，因此有成为新型药物的潜力。本研究采用毕赤酵母真核表达系统来生物合成抗菌肽Odorgrin A和Odorgrin C，为大量获取抗菌肽资源提供技术支撑。 方法：依照Odorgrin A和C的氨基酸序列、采用酵母偏爱密码子分别设计并化学合成了相应的目的基因序列。目的片段从合成质粒上用Xho Ι和EcoR Ι双酶切下后，与经同样限制酶完全酶切pPIC9K载体所获得的两个大片段直接连接，并转化至大肠杆菌DH5α。用PCR扩增、酶切及测序检测，鉴定正确的重组质粒。提取大量表达载体pPIC9K - Odo A和C并使之线性化后经电击法分别转化毕赤酵母（Pichia pastoris）GS115宿主菌，用营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序检测，鉴定并筛选出对G418具高抗性的Odorgrin A和C重组酵母菌。用甲醇对之进行诱导表达，SDS - PAGE电泳及反相层析检测表达产物，并做抑菌活性检测。 成果：PCR扩增、酶切及测序等结果表明表达载体pPIC9K - Odo A和C构建成功。营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序等证实pPIC9K - Odo A和C已整合入酵母基因组中。SDS - PAGE电泳及反相层析结果表明抗菌肽Odorgrin A和C成功地获得了分泌表达。而抑菌活性实验则检测到部分阳性克隆菌诱导分泌表达的抗菌肽Odorgrin A和C都对测试菌的生长具有较高（>94%）的抑制率。 结论：无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因的表达载体都构建成功，并且都在毕赤酵母系统中获得了成功表达。 Background ＆ Objective: In the recent twenty years, a lot of pathogenic bacteria have come forth in clinic with durable trait derived from making use of and abusing the traditional antibiotics. Therefore, studying antimicrobial peptides, not be easy to be invalidated by durable bacteria, are becomimg popular and important. The skin antimicrobial peptides of Odorrana grahami with broad spectrum antibacterial activity and no toxicity to eukaryotic cell, discovered by previous research work of our workgroup, are looked forward to being potential medication. Pichia pastoris expressional system was used for biosynthesis antimicrobial peptides Odorgrin A and Odorgrin C in this study, for producing abundant antimicrobial peptides. Methods: The foreign fragments which included Odorgrin A or Odorgrin C gene according to their amino acid sequence respectively were synthesized based on the biased codon usage of yeast. The DNA fragments, obtained from the plasmids containing them by digested with Xho Ι and EcoR Ι, were directly ligated with the two bigger fragments obtained from the vector pPIC9K by digested with the same restriction enzymes. And then they were transformed into Escherichia coli DH5α to be selected and amplified positive colonies. The recombinants were testified by using PCR amplification, enzymes digestion and sequencing of the foreign fragment. After the expressional vector pPIC9K - Odo A and pPIC9K - Odo C were linearized, they were transformed into Pichia pastoris GS115 strain by the electroporation. Then the positive colonies which were of the highest geneticin resistant were selected through auxotrophic screening, genetic resistant screening, PCR amplification and sequencing of the inserted fragment. Methanol was used to induce the recombinant yeasts to express the foreign gene. SDS-PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment were used to testify the expressional products. Results: The evidences of PCR, enzymes digestion and sequence analysis confirmed that the expressional vector pPIC9K - Odo A and pPIC9K - Odo C have been constructed correctly. The results of auxotrophic screening, of genetic resistant screening, of PCR and sequencing of the foreign fragment showed that Odorgrin A and Odorgrin C gene have been homologous integrated with the Pichia pastoris genome. And it was also testified that antimicrobial peptides Odorgrin A and Odorgrin C have been expressed successfully by using SDS - PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment. Conclusion: The expressional vector of the skin antimicrobial peptides Odorgrin A and Odorgrin C gene of Odorrana grahami have been constructed correctly and both of the genes have been expressed successfully in Pichia pastoris system in this study.

一株掷孢酵母利用红薯淀粉酶解液积累油脂研究

本论文以红薯淀粉的双酶法水解液为碳源，从19 株红色酵母中筛选出一株油脂含量较高的菌株掷孢酵母（Sporobolomyces reseus）As.2.618。为了提高掷孢酵母（S.reseus）As.2.618 的油脂产量，考察了培养基组成对该菌生长情况及油脂积累的影响。用均匀设计法对培养基组成进行了优化，由DPS软件得出的优化结果为：还原糖103g/L、酵母粉11.5g/L、磷酸二氢钾0.3g/L、硫酸镁0.15g/L。生物量可达19.23 g/L,油脂含量为3.875 g/L。研究了添加二价离子对该菌的生长及油脂积累的影响,结果表明Zn2+对该菌生长和油脂积累都有显著促进作用。研究了发酵条件以及添加氧载体正十二烷对该菌发酵的影响，表明添加正十二烷有利用于该菌生长与油脂积累。得出最佳发酵条件是：在还原糖103g/L、酵母粉11.5g/L、磷酸二氢钾0.3g/L、硫酸镁0.15g/L。添加30mg/L 硫酸锌,接种量为5％，在24h 后添加2g/L 的碳酸钙和2%(v/v)正十二烷，pH6.0 培养温度为27℃，转速为200r/min，培养时间为7 天的条件下，该菌生物量干重可达35.05g/L,油脂含量也达11.98g/L。Lipid is one of the basic material for life-sustaining activities andimportant industrial materials. As lipid resources mainly come from the animal andthe plant, the problem of lipid lack is encountered at times. The lipid frommicroorganisms is the substitute and superior to the above lipid with a short period ofproduction and much cheaper fermentation materials such as agricultural and sidelineproducts or wastes of crop.Thus large scale production and broad application ofmicrobial lipid will be efficient not only in substitute of the animal and the plant lipidfor food and industrial field , but also inducing a new way leading to solve the energyproblem.For the purpose of exploring the characteristics of lipid production of redyeasts from sweet potato starch hydrolysates. 19 red yeasts are screened for thecapability of lipid producing and one strain Sporobolomyces reseus As.2.618 withsuperior performance is sellected.To improve the Sporobolomyces reseus As.2.618’s capability of lipidaccumulation , the components of the medium, which may influence the growth of thestrain and the lipid yield have been studied. To get the optimum mediumcomponents ,the “uniform design” was used .The DPS software gave the optimummedium component is: reducing sugar 103 g/L、yeast extract 11.5 g/L、KH2PO4 0.3g/L、MgSO4 0.15 g/L. The biomass could reach up to 19.23 g/L and lipid yield 3.87g/L with the above composition of fermentation medium.Furthermore the fermentation conditions , addition of the divalent metal ionsand the oxygen vector to increase the strain’s lipid producing capability are tested.The optimum condition is : reducing sugar 103 g/L、yeast extract 11.5 g/L、KH2PO40.3 g/L、MgSO4 0.15 g/L，Adding 30mg/L ZnSO4,and adding 2g/L CaCO3 2％(v/v)n-dodecane after 24h’s fermentation. the optimal fermentation condition were asfollow :30ml medium in the 500ml flask with initial pH 6.0,the flasks with 5%inoculation volume were at 200r/min shaking speed for 7d’s fermentation at27 .Under this kind of condition the high biom ¡æ ass which reach to 35.05 g/L could begot ,the yield of lipid also could reach to 11.98g/L.

固定化红酵母细胞合成L-苯丙氨酸研究

In this paper, bioconversion of trans-cinnamic acid(t-Ca)to L-phenylalanine (L-phe) has been investigated by using immobilized yeast cells with induced L-phe Ammonia-lyase(PAL, EC.4.3.1.5) as biocatalysts. The contents are the following. (1) Thirty strains of yeasts, including two genera (Rhodotorula, Sporobolomyces), six species (R. glutinis R. minuta,R.rubra,R.sineses,R.roseus and S.salmonicolor)were screened for their ability to converse the substrates, t-Ca and ammonia, to the product, L-phe, by using yeast cells as biocatalyst, and primary evaluation for PAL activity of the selected strains was investigated. From the results of the screening experiments, it was found that 22 strains were able to produce L-phe from t-Ca with the range of conversion yield from 2% to 67%. Studies on PAL formation time course during cultivation show that the maximum PAL activity of several different strains ranges from 2.3 to 14.4×10-3U/mg cell dry weight. The biomass of tested strains at their maximum enzyme activity is also greatly varied. (2)One of the selected strains, R. rubra as 2.166, was used for immobilized cells as biocatalysts to produce L-phe. The optimum conversion conditions and effective stablization agents were investigated. The results shown that polyacrylamide gel was chosen as a suitable matrix for immobilization of the yeast cells, and it can retain 88% of the PAL activity in the reverse direction at the following reactive conditions: [t-Ca]: 34mM. [NH4OH]: 6.OM.PH10.00, temperature: 30℃. (3) The effects of various kinds of effectors on the production of L-phe were also examined. Membrane permeabilizing agents can stimulate L-phe synthesis, but make the stability of PAL decline greatly. Polyalchoholic agents and glutamic acid were very effective for the stabilization of PAL. At the presence of glutamic acid (5%), the half life of L-phe productivity with the immobilized cells was extended to 192 hours, which was much higher than most of that having been reproted, while the half life of resting cells was only about 15 hours. (4) Use of initial velocity studies on the kinetics of enzyme-catalized reaction indicated that the apparent Km value was 13.0mM for the immobilized cells, and 4.8mM for the resting cells. Thermostability of the immobilized cells was better than the resting cells. Fluid bed bioreactor is more effective than batch bioreator in prolonging the thermostability of the biocatalysts. (5) CGA- 688 resin column chromatographic procedure was employed in the isolation and purification of L-phe, t-Ca and other substances from the reactire mixture. (6) Preparative-scale production of L-phe on a level of gram amount by immobilized cells from the culture broth of R. rubra AS2.166 allowed for the conversion yield with 30%. The characteristic physico-chemical criteria (including melting point, optical activity, elements analysis, IR, NMR) are the same with the standard L-phe. 本文报告了利用诱导的苯丙氨酸解氨酶 (PAL.EC.4.3.1.5)催化反式肉桂酸(t-Ca)氨加 成制备L-苯丙氨酸(L-phe)的研究，主要内容为：(1) 我们搜集了三十株酵母菌株，利用全细胞转化t-Ca生成L-phe的能力进行了直 接筛选，并对其PAL活性水平进行了初步评估研究。研究结果表明，其中22株酵母具有转化t-Ca生产L-phe的能力，它们包括 Rhodotorula glutinis,R.rubra, R.sineses 和Sporobolomyces roseus 的菌株，转化率在2-67%。细胞生长和PAL形成过程的研究 表明，不同菌株PAL最大活力在2.3-14.4×10-3U/mg 细胞干重，达到最大PAL活性时各株酵母的生长情况也极不一致。(2) 利用筛 选出的一株深红酵母R.rubra AS2.166 作为供试菌株，研究了细胞固定化条件下生物转化的最适条件及PAL在固定化条件下的稳定 性。结果表明以聚丙烯酰胺凝胶包埋法较为理想，能使细胞合成L-phe活力保持88%，最适t-Ca浓度为34mM，最适NH4OH浓度为6M,最 适PH10.0，最适温度45℃。(3) 多种效应物对L-phe 合成的影响研究表明：表面活性剂能刺激L-phe的合成，但使PAL稳定性下降。 多羟基化合物及Glu对PAL的稳定十分有效在有Glu存在下，能使固定化细胞合成L-phe的半寿期达192小时左右，高于大部分现已报 导的固定化结果。(4) 用初速度法研究了深红酵母AS2.166中PAL的酶促反应特征，测得固定化细胞对t-Ca的表观米氏常数Km为 13.0mM，全细胞为4.8mM，细胞固定后热稳定性提高。(5) 建立了适合低浓度分离纯化产物与底物的聚苯乙烯大孔树脂柱层析技术 ，能使L-phe与t-Ca及产物混合物中其它成分有效分开。(6) 利用固定化的R.rubra AS2.166细胞所做的制备实验能够使L-phe的产 率达到30%左右，其主要的理化指标(包括熔点、比旋光度、元素分析、IR、NMR等)与标准L-phe一致。

泸州老窖古酿酒作坊空气微生物分布特征研究

本文主要研究了泸州老窖古酿酒作坊内外环境空气真菌和空气细菌的群落结构和分布特征。结果如下： 作坊内外环境空气微生物浓度差别显著，并随季节变换而变化，春、夏季微生物浓度较高，秋、冬季较低，空气真菌在夏季达到最高，细菌在春季最高。 古作坊内外环境检测到的真菌均为16 属，但优势菌属不同，作坊外的优势菌属为青霉属(Penicillium)、曲霉属(Aspergillus)、无孢菌(non-sporing)、枝孢霉属(Cladosporium)和链格孢属(Alternaria)；而作坊内优势菌属为曲霉属、青霉属、酵母菌(Yeast)、无孢菌，作坊内还含有较高浓度的根霉属（Rhizopus）、毛霉属（Mucor）、短梗霉属（Aureobasidiu），枝孢霉属和链格孢属等，曲霉属、酵母菌、根霉属、毛霉属为古酿酒作坊重要的酿酒真菌，青霉属、链格孢属为酿酒不利菌群。对古作坊内曲霉属进行了初步鉴定，主要是小冠曲霉（A.cristatellus）、米曲霉（A.oryzae）、黑曲霉（A.niger）和白曲霉（A.cadidus）。 空气细菌10 属21 种，作坊内外环境的优势菌属均为芽孢杆菌属（Bacillus）、微球菌属(Micrococcus)、葡萄球菌属(Staphylococcus)、假单胞菌属(Pseudomonad)，其中芽孢杆菌属在作坊内占有绝对的优势，浓度比在40℅以上，是古酿酒作坊重要的酿酒细菌，另外还检测到较高浓度的乳酸杆菌（lactobucillus），这类菌容易使酒味发涩发苦，为酿酒不利菌。 作坊内外环境空气微生物表现出明显的交流现象。作坊内，青霉属、枝孢霉属、链格孢属、葡萄球菌属等杂菌占有一定比例；而在作坊外,芽孢杆菌属、曲霉属、根霉属(Rhizopus)、酵母菌等处于相对较高水平，绿化环境较好的营沟头作坊内的短梗霉属，枝孢霉属和链格孢属等杂菌含量低于什字头和新街子作坊。 The community structure and distribution characteristic of airborne microbes was investigated in ancient brewage workshops of luzhoulaojiao. The results are as follows: The concentration of airborne microbes was different in interior and exterior environment of ancient workshops, and also varied by seasons. microbial concentration was higher in spring and summer, and lower in fall and winner. The highest levels of airborne bacteria was in spring, but the fungal’s in summer. The identified genus of fungi were 16 in interior and exterior environment of the ancient workshops. But the dominant genus were different , The advantage genus in the interior were Aspergillus, Yeasts, Penicillum and Nonsporing and in the exterior were Penicillum, Nonsporing, Cladosporium, Aspergillus and Aureobasidiu. Rhizopus ,mucor, Aureobasidiu, Cladosporium, Alternaria and all also were at a higher level. Among these, Aspergillus, Yeasts, Rhizopus ,mucor are important vintage flora . Penicillum, Alternaria do harm to vintage. Aspergillus of ancient workshops was identified , the preponderant aspergillus species were A.cristatellus, A.oryzae, A.niger and A.cadidus in ancient brewage workshops. 10 genus 21 species bacteria were identified, the advantage genuses among the interior and exterior of the three workshops were bacillus, microccus, Staphylococcus Pseudomonas. Bacillus, which account for beyond 40℅ of the total bacteria concentration in all sampling pots, was the most dominant genus. Lactobacillus was identified at a high level in ancient workshops, it makes spirit taste bitter and astringent. So it is not a kind of good bacterium for vintage. The fungus in the interior and exterior atmosphere characterized intercommunion phenomenon. Obviously, the concentration of profitless fungus such as Penicillum, Cladosporium, Alternaria appeared in the interior, and the fungus such as Bacillus, Aspergillus, Rhizopus and Yeasts in the exterior were at a relatively high level. the harmfull fungus in yinggoutou workshops such as Aureobasidiu, Cladosporium, Alternaria and all were lower than shenzitou and xinjiezi workshops.