Transcriptome and proteome analysis of xylose-metabolising Saccharomyces cerevisiae

Increasing concern about global climate warming has accelerated research into renewable energy sources that could replace fossil petroleum-based fuels and materials. Bioethanol production from cellulosic biomass by fermentation with baker s yeast Saccharomyces cerevisiae is one of the most studied areas in this field. The focus has been on metabolic engineering of S. cerevisiae for utilisation of the pentose sugars, in particular D-xylose that is abundant in the hemicellulose fraction of biomass. Introduction of a heterologous xylose-utilisation pathway into S. cerevisiae enables xylose fermentation, but ethanol yield and productivity do not reach the theoretical level. In the present study, transcription, proteome and metabolic flux analyses of recombinant xylose-utilising S. cerevisiae expressing the genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis and the endogenous xylulokinase were carried out to characterise the global cellular responses to metabolism of xylose. The aim of these studies was to find novel ways to engineer cells for improved xylose fermentation. The analyses were carried out from cells grown on xylose and glucose both in batch and chemostat cultures. A particularly interesting observation was that several proteins had post-translationally modified forms with different abundance in cells grown on xylose and glucose. Hexokinase 2, glucokinase and both enolase isoenzymes 1 and 2 were phosphorylated differently on the two different carbon sources studied. This suggests that phosphorylation of glycolytic enzymes may be a yet poorly understood means to modulate their activity or function. The results also showed that metabolism of xylose affected the gene expression and abundance of proteins in pathways leading to acetyl-CoA synthesis and altered the metabolic fluxes in these pathways. Additionally, the analyses showed increased expression and abundance of several other genes and proteins involved in cellular redox reactions (e.g. aldo-ketoreductase Gcy1p and 6-phosphogluconate dehydrogenase) in cells grown on xylose. Metabolic flux analysis indicated increased NADPH-generating flux through the oxidative part of the pentose phosphate pathway in cells grown on xylose. The most importantly, results indicated that xylose was not able to repress to the same extent as glucose the genes of the tricarboxylic acid and glyoxylate cycles, gluconeogenesis and some other genes involved in the metabolism of respiratory carbon sources. This suggests that xylose is not recognised as a fully fermentative carbon source by the recombinant S. cerevisiae that may be one of the major reasons for the suboptimal fermentation of xylose. The regulatory network for carbon source recognition and catabolite repression is complex and its functions are only partly known. Consequently, multiple genetic modifications and also random approaches would probably be required if these pathways were to be modified for further improvement of xylose fermentation by recombinant S. cerevisiae strains.

Translational fusion of two beta-subunits of human chorionic gonadotropin results in production of a novel antagonist of

The strategy of translationally fusing the alpha-and beta-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active singlechain analog hCG alpha beta expressed using Pichia expression system. Using the same expression system, another analog, in which the alpha-subunit was replaced with the second beta-subunit, was expressed (hCG beta beta) and purified. hCG beta beta could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose- dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCG alpha beta and hCG beta beta using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCG beta beta interacts with two LH receptor molecules. These studies demonstrate that the presence of the second beta-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of alpha-subunit, the molecule is unable to elicit response. The strategy of fusing two beta-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

Hybrid molecules of carvacrol and benzoyl urea/thiourea with potential applications in agriculture and medicine

Benzoyl phenyl urea, a class of insect growth regulator's acts by inhibiting chitin synthesis. Carvacrol, a naturally occurring monoterpenoid is an effective antifungal agent. We have structurally modified carvacrol (2-methyl-5-1-methylethyl] phenol) by introducing benzoylphenyl urea linkage. Two series of benzoylcarvacryl thiourea (BCTU, 4a-f) and benzoylcarvacryl urea (BCU, 5a-f) derivatives were prepared and characterized by elemental analysis, IR, H-1 and C-13 NMR and Mass spectroscopy. Derivatives 4b, 4d, 4e, 4f and 5d, 5f showed comparable insecticidal activity with the standard BPU lufenuron against Dysdercus koenigii. BCTU derivatives 4c, 4e and BCU 5c showed good antifungal activity against phytopathogenic fungi viz. Magnaporthe grisae, Fusarium oxysporum, Dreschlera oryzae; food spoilage yeasts viz. Debaromyces hansenii, Pichia membranifaciens; and human pathogens viz. Candida albicans and Cryptococcus neoformans. Compounds 5d, 5e and 5f showed potent activity against human pathogens. Moderate and selective activity was observed for other compounds. All the synthesized compounds were non-haemolytic. These compounds have potential application in agriculture and medicine. (C) 2012 Elsevier Ltd. All rights reserved.

[Discurso do Deputado Vasco dos Reis : Sessão da Câmara dos Deputados de 20 de janeiro de 1948]

Destaca a importância da interiorização da capital do país, como forma de conter os movimentos migratórios para os centros litorâneos e a decadência das atividades agrícolas e pastoris em diversas unidades federativas.

果实采后病害生物防治及其机理研究

　　分离和筛选了5种能有效防治采后果实病害的拮抗菌。其中，季也蒙假丝酵母(Candida guiliermondii(Cast) Langeroret Guerra)从引种拮抗菌中筛选获得，枯草芽孢杆菌（Bacillus subtilis）B-912从土壤中分离筛选获得，膜醭毕赤酵母（Pichia membranefaciens hansen）从桃果实伤口上分离获得，隐球酵母(Cryptococcus albidus (Saito) Skinner)和丝孢酵母（Trichosporon sp.）从桃果实表面分离获得。本文主要研究了这些拮抗菌对桃、油桃、苹果、梨和柑桔等我国主要水果采后病害的防治效果，并对其可能的抑菌机理进行了初步研究。结果如下： 1. Sx108 CFU/mL的C．guiliermondii和P．membranefaciens悬浮液可完全抑制病菌孢子浓度为5x104个/mL时桃和油桃果实软腐病(Rhizopus stolonifer(Ehrenb.ex Fr.) Vuill.)在25℃，15℃和3℃下的发生。lx108 CFU/mL的C．albidus和Trichosporon sp.悬浮液可完全抑制孢子浓度分别为lx105个/mL和5x104个/mL时苹果灰霉病(Botrytis cinerea)和青霉病（Penicillum expansum）在23℃-25℃和1℃下的发生。C．albidus和Trichosporon sp.对梨灰、青霉病也有一定抑制效果。B-912对柑桔果实青霉病(Penicillium italicum)、绿霉病(Penicillium digitatum)和核果类果实褐腐病(Monilinia fructicola)也有极好的抑制效果。生物防治效果与拮抗菌的浓度成正比，与病菌孢子浓度成反比。 2．拮抗酵母菌在室温和冷藏条件下都能迅速在果实伤口定殖，接种酵母菌48 h后，数量可增加20倍以上。拮抗菌和病菌孢子的接种时间与生物防治效果有关，先接种拈抗菌的抑菌效果显著地好于同时或后于病菌接种的效果。 3．温度对拮抗酵母菌的抑菌活力没有明显影响，无论是在室温还是在冷藏条件下，拮抗酵母菌都有同样的抑菌效果。但拮抗细菌B-912的抑菌效果与温度有一定关系。较高的温度有利于细菌拮抗作用的发挥。 4．拮抗菌能与常规的果实采后处理措施如钙处理、化学杀菌剂、冷藏和气调贮藏相结合。酵母菌与2% CaC12配合能明显地增强其抑菌能力;拮抗菌与低浓度的杀菌剂如扑海因混合使用，可达到高浓度杀菌剂的抑病效果;C．albidus和Trichosporon sp.对果实采后气调贮藏环境有良好的适应性，它们在气调下对采后苹果、梨的灰霉病和青霉病的抑制效果比冷藏条件下的好。 5．细菌B-912的抑菌机理与其产生抗菌素有关，B-912的滤液在in vitro上能有效地抑制病菌孢子的萌发，在invivo上也能明显地抑制果实采后病害的发生。拮抗酵母菌的抑菌机理则较复杂，但主要与病菌竞争营养有关，同时，C．guilliermondii和P．membranefaciens对软腐菌的抑制还通过产生水解酶如β-1，3一葡聚糖酶和几丁酶与病原菌直接作用，并参与诱导寄主产生抗性等

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。

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。

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。

水稻2,3-氧化鲨烯环化酶基因的功能研究

植物通过异戊二烯代谢途径合成多种具有生物活性和功能的三萜及甾醇类化合物，它们在调节植物生长发育、维持膜的完整和功能、抵抗病原微生物侵染中发挥着重要的作用。2,3-氧化鲨烯为三萜和甾醇合成途径的分枝点，参与这一关键步骤的酶被通称为2,3-氧化鲨烯环化酶（OSCs）。本研究系统分了水稻基因组中全部11个OSC基因序列，发现其中四个可能为假基因。亚种间非同义替换率Ka和同义替换率Ks的比值（Ka/Ks）以及进化树的分析表明OsOSC8是单子叶植物特有的功能保守基因，而OsOSC9在水稻两个亚种间发生了功能快速进化，这种快速进化的基因往往参与植物和病原菌相互作用的代谢途径。 根据基因结构、表达谱以及与其它植物已知功能的OSC酶氨基酸序列的比对推测OsOSC3可能具有环阿屯醇合成酶的功能，参与植物甾醇的合成，而OsOSC7、OsOSC10和OsOSC11可能具有β-香树素合成酶的功能，其余OSCs可能参与合成其它三萜化合物。为了进一步分析和验证OSCs酶的功能，将水稻7个OSC基因的开放阅读框（ORF）构建到酵母表达载体并在pichia酵母中表达，发现仅有OsOSC9和OsOSC12能够将酵母内源的2,3-氧化鲨烯分别环化为四环三萜化合物Parkeol和植物中稀有的五环三萜化合物Isoarborinol，目前还未在其它植物中发现参与这两种三萜化合物的基因。另外，水稻所有的OSC基因均不能互补酵母羊毛甾醇缺陷型菌株，表明水稻OSCs不具有合成羊毛甾醇的功能。 RNAi沉默以及启动子融合GUS的表达实验发现OsOSC8可能参与花粉的发育，该基因的下调影响水稻的育性，暗示水稻中存在一个可能与雄性不育有关的三萜代谢途径。水稻其它OSC基因RNAi植株可能在逆境环境和病原菌侵染下才会显现出表型。

Feasibility study of biofuel production from freshwater fern, Azolla sp in Anzali Wetland

Production of bioethanol through acidic and enzymatic hydrolysis of aquatic Azolla sp., as a new source of bio-mass, has been performed, as a means to control increasing growth and reducing undesirable effects of this plant in Anzali lagoon. After sampling, drying and crushing, Azolla was hydrolyzed, using diluted acid and enzyme. Diluted acid hydrolysis was done using both autoclave and a high-pressure system (Batch Synth® Microwave synthesizer). The effects of temperature and time (in autoclave) and concentration of acid (in both) were compared. Cellubrix®, a ommercial cellulase source, was used for enzymatic hydrolysis process. The amounts of reducing sugars, glucose and furfural, released from hydrolyzate, were measured. To produce alcohol, Sacchromyces cerevisiae (to ferment sixcarbon sugars), Zygowilliopsis californica and Pichia stipitis (to ferment five-carbon and sixcarbon sugars) were used. Maximum amounts of glucose (4.83% w/w) and reducing sugars (14.15% w/w) were obtained using acid hydrolysis in autoclave. In the microwave oven, maximum glucose (5.04% w/w) and reducing sugars (13.27 w/w) were obtained at 180 and 200 °C, respectively. Under these conditions, maximum produced furfural was 1.54 g/L. The difference between amounts of furfural obtained from acid hydrolysis of Azolla in microwave oven compared to autoclave was statistically significant. Amounts of alcohol produced and its yields were 3.99 g/L and 33.13% for S. cerevisiae in 48 hours, 3.73 g/L and 30.45% for Pichia stipites in 48 hours, and 3.73 g/L and 30.45% for Z. californica in 24 hours after inoculation, respectively, with significant differences. Statistical comparison of results showed significant differences (P<0.05) in glucose production, at different conditions. Amounts of reducing sugars and glucose increased after optimization of levels of acid, time, and temperature. The overall optimum released sugar and glucose were obtained with 1.67% (w/v) acid using autoclave. Higher temperatures in microwave oven caused a significant increase (P<0.05) in furfural. Furfural severely inhibits fermentation. Hence, regarding the issues of energy consumption and time, amounts of inhibiting substances and sugar production, autoclave is found to be superior to the high temperature and pressure, generated in microwave oven, for hydrolyzing Azolla. Furthermore, given the amounts of Azolla in Anzali lagoon, it may be recommendable to use this plant as a biomass resource.

Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis

P>NF-kappa B is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-kappa B-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor kappa B (I kappa B)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-kappa Bs and phylogenetic analysis suggested that EsRelish was a member of the NF-kappa B family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.

海北站周围3种外来物种入侵状况的初步研究

通过对海北生态系统定位站附近3种植物播娘蒿Descurainia sophia、荠菜Capsella bursa-pastoris、灰绿藜Chenopodium glaucum的分布情况的调查,初步分析了3种植物的扩散策略,并预测了其对高寒草甸生态系统潜在的影响.

Sistemas agrícolas sustentáveis para regiões semi-áridas.

Resumo: A maioria dos sistemas de produção em prática no trópico semi-árido brasileiro pela agricultura familiar apresenta baixa ou nenhuma sustentabilidade, devido ao uso de tecnologias quase sempre agressivas ao ecossistema. Como consequência, observa-se, nos sertões nordestinos degradação ambiental generalizada, baixa produtividade das atividades agrícolas, pastoris e madeireiras, inviabilização da agricultura, queda da qualidade devida e êxodo rural acentuado. Urge, pois, que se forneça ao agricultor alternativas de sistemas de produção sustentáveis que atendam a quatro objetivos, quais sejam, a fixação da agricultura migratória, a adequação do manejo pastoril, a racionalização da produção de madeira e a uma forte integração dessas atividades. Para tanto, deve-se: excluir as queimadas, substituir o desmatamento pelo raleamento da vegetação lenhosa, manter um aporte substancial de matéria orgânica, utilizar Ieguminosas como fonte de adubo verde, ajustar a carga animal para a capacidade de suporte da área e adotar-se o corte seletivo e manejo da rebrota na exploração madeireira. A adoção dessas tecnologias na certa contribuirá: para a viabilização ecológica, social e econômica da agricultura familiar nos serões nordestinos, para a sustação dos processos de degradação ambiental, para a melhoria da qualidade de vida da população e para a redução do êxodo rural.