Artificial interspecific hybridization between Macrobrachium species

Viable F-1 hybrids were obtained from crosses of female Macrobrachium nipponense and male Macrobrachium hainanense involving spermatophore transfer and artificial insemination. This represents the first successful known case of hybridization of two Macrobrachium species by means of artificial insemination. The hatching rate was over 90%. About 20-60% of newly hatched larvae metamorphosed to postlarvae. The morphological characteristics of the hybrids resembled a combination of features of both parents. Malate dehydrogenase (MDH) and esterase (EST) isozyme electrophoresis indicated parents and F-1 hybrids showed co-dominant expression of the paternal and maternal alleles controlling the isozymes and confirmed the hybridization. (C) 2004 Elsevier B.V. All rights reserved.

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.

Characterization of an iridovirus from the cultured pig frog Rana grylio with lethal syndrome

Three virus isolates, RGV-9506, RGV-9807 and RGV-9808, were obtained from cultured pig frogs Rana grylio undergoing lethal infections. Previously, the first isolate, RGV-9506, was shown to be an iridovirus based on ultrastructural and morphological studies. In the present study, the original isolate, along with 2 recent ones, were more extensively characterized by experimental infection studies, histopathology, electron microscopy, serological reactivity, gel electrophoresis of viral polypeptides and DNA restriction fragments, PCR amplification, and nucleic acid sequence analysis of the major capsid protein (MCP) gene. The 3 isolates were shown to be identical to each other, and very similar to FV3, the type species of the genus Ranavirus (family Iridoviridae). These results suggest that RGV should be considered a strain of FV3, and indicate that FV3-like iridoviruses are capable of causing widespread, severe disease among cultured frogs.

A detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription polymerase chain reaction

A rapid, sensitive and highly specific detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription-polymerase chain reaction (RT-PCR) has been developed. Two pairs of PCR primers were synthesized according to the cloned cDNA sequences of the GCHV-861 strain. For each primer combination, only one specific major product was obtained when amplification was performed by using the genomic dsRNA of GCHV-861 strain. The lengths of their expected products were 320 and 223 bp, respectively. No products were obtained when nucleic acids other than GCHV-861 genomic RNA were used as RT-PCR templates. To assess the sensitivity of the method, dilutions of purified GCHV-861 dsRNA total genome (0.01 pg up to 1000 pg) were amplified and quantities of as little as 0.1 pg of purified dsRNA were detectable when the amplification product was analyzed by 1.5% agarose gel electrophoresis. This technique could detect GCHV-861 not only in infected cell culture fluids, but also in infected grass carp Ctenopharyngodon idellus and rare minnow Gobiocypris rarus with or without hemorrhagic symptoms. The results show that the RT-PCR amplification method is useful for the direct detection of GCHV.

Continuous-flow polymerase chain reaction microfluidics by using spiral capillary channel embedded on copper

This paper presents a novel method for performing polymerase chain reaction (PCR) amplification by using spiral channel fabricated on copper where a transparent polytetrafluoroethylene ( PTFE) capillary tube was embedded. The channel with 25 PCR cycles was gradually developed in a spiral manner from inner to outer. The durations of PCR mixture at the denaturation, annealing and extension zones were gradually lengthened at a given flow rate, which may benefit continuous-flow PCR amplification as the synthesis ability of the Taq polymerase enzyme usually weakens with PCR time. Successful continuous-flow amplification of DNA fragments has been demonstrated. The PCR products of 249, 500 and 982 bp fragments could be obviously observed when the flow rates of PCR mixture were 7.5, 7.5 and 3.0 mm s(-1), respectively, and the required amplification times were about 25, 25, and 62 min, respectively. Besides, the successful segmented-flow PCR of three samples ( 249, 500 and 982 bp) has also been reported, which demonstrates the present continuous-flow PCR microfluidics can be developed for high-throughput genetic analysis.

毛细管电泳电化学检测联用的应用研究

本论文探讨了毛细管电泳电化学检测器的应用。主要包括以下六个方面的内容：1．毛细管电泳与电化学检测联用测定别嗓呤醇及其代谢产物氧化镖吟醇。该方法简单、快速、灵敏。并且应用于加标尿样的分析时也取得了令人满意的结果。2．采用毛细管电泳一安培检测来定量测定中草药中的活性组分姜黄素，用自制的磷酸三丁酷树脂萃取柱来预处理样品，同时实现对姜黄素的浓缩。经毛细管电泳分离后，姜黄素可以用碳纤维电极检测。3．毛细管电泳电化学发光对尿液中的丙环定的检测。用离子交换柱尽可能除去尿中的离子的干扰，电化学发光试剂印比淀钉采用柱后加入的方式，具有三级胺结构的丙环定可被定量检测到。4．溶胶一凝胶一碳复合材料电极作为毛细管电泳安培检测器的表征。我们报道了这种电极的制作，探讨了不同尺寸的电极在毛细管电泳中的应用。当电极直径100μm，一些分析指标如：检测限、线性范围以及重现性都较好，可以做为安培检测器与毛细管电泳联用。5，毛细管电泳脉冲安培检测生物胺。分离并定量检测了四种生物胺，腐胺、尸胺、亚精胺及精胺。并用该方法测定了牛奶中的生物胺。该方法比已报道的毛细管电泳间接紫外检测的检测限低，比气相色谱及液相色谱需样品体积小，不需要烦琐的衍生步骤。6．溶胶一凝胶一碳复合材料电极作为电泳芯片的安培检测器。我们报道了一种简单，重现性好的芯片上的盘电极的制作方法。由于该安培检测器最大的特点就是稳定性好，重现性高所以我们在其上又沉积了一层铜，以扩大该芯片的应用范围。用集成了该安培检测器的芯片，我们分别测定了肾上腺素和葡萄糖。

ITO导电玻璃和PDMS微芯片毛细管电泳电化学发光检测的研究

微全分析系统是目前很前沿的研究领域，尽管现在还没有真正意义的微全分析系统出现，但它代表了分析科学的发展趋势。本文主要研究了ITO导电玻璃和PDMS微芯片毛细管电泳和电化学发光检测方法。微芯片毛细管电泳对与其联用的检测器有相当高的要求，一些传统的检测方法很难适应于微芯片毛细管电泳。电化学发光检测是一种新兴的检测技术，在化学、生物、医学诊断以及免疫分析中展现出良好的应用前景。如何实现和完善微芯片毛细管电泳与电化学发光检测联用技术是本论文的重点。我们采用聚二甲基硅氧烷（poly（dimethylsiloxone），简称PDMS）和玻璃作为芯片材料，以锢锡氧化物（indium桩n oXide，简称工T0）导电玻璃为工作电极设计了一种集成化的微芯片毛细管电泳电化学发光检测器。其中，芯片的底片由工TO导电玻璃经光刻、化学腐蚀等方法处理后得到。ITO是一种透明的导电材料，作为工作电极集成到芯片的底片上，PDMS层与芯片底片采用可逆键合的方式键合，大大简化了操作并提高了电化学发光信号的采集效率。我们采用脯氨酸作为被测物对检测器进行了表征。在实验过程中，微芯片毛细管电泳及工T0工作电极都表现出良好的稳定性。我们还提出了电化学和电化学发光同时检测技术，应用于微芯片毛细管电泳和常规毛细管电泳。在这种电化学和电化学发光双检测模式中，三联吡陡钉（Ru（bpy）32+既作为电化学发光检测所需的发光试剂与被分析物反应生成激发态的Ru（bpy）32+＊产生电化学发光信号，又在电极表面平行催化电化学反应得到增强的电流响应，提高电化学检测的灵敏度。电化学信号与电化学发光信号同时产生并分别记录，从而实现了电化学和电化学发光同时检测。我们将这种检测技术与芯片或常规毛细管电泳结合，以多巴胺及三种药物分子山蓖若碱、氧氟沙星和利多卡因作为被测物对其进行了表征。这种同时检测方法与其它多检测模式相比更为简单、方便，比单一的电化学或电化学发光检测可以获得更多的被分析物信息，扩大单一检测方式的应用范围。

石油污染土壤微生物多样性分析及芘降解菌的鉴定

石油污染是目前最严重的环境问题之一，我国有近千万亩的耕地受到程度不同的石油污染。沈抚灌区位于辽宁省沈阳市和抚顺市之间，全长70多千米，是我国最大的石油污染灌区之一，有四五十年的污灌历史，已对当地的生态环境造成了严重的影响。随着人们对污水灌溉危害的认识，从20世纪80年代起，逐步停止了污水灌溉，开始了清水灌溉和改为旱田耕作，但污染问题仍然存在。目前，由于对该地区污染土壤微生物类群、原位降解菌及它们对环境条件变化的响应等信息缺乏了解，限制了对该地区污染土壤的生物修复。 分子生物学研究手段及稳定同位素分析技术，为污染土壤微生物生态学研究提供了全新的研究手段，为突破当前石油污染土壤生物修复研究止步不前的局面指引了方向。本论文采用变性梯度凝胶电泳（Denaturing Gradient Gel Electrophoresis, DGGE）和磷脂脂肪酸分析（Phospholipid Fatty-acid Analysis, PLFA）分析技术，对沈抚灌区水改旱田石油污染土壤的微生物多样性与环境指标的相关规律进行了分析；以PLFA为生物标示物，结合气相色谱-稳定同位素比率质谱（Gas Chromatography Combustion Isotope Ratio Mass Spectrometry, GC-c-IRMS）分析技术，对石油污染土壤和相对清洁土壤中多环芳烃菲和芘的土著降解菌群进行了鉴定；并以不同的吸附性载体为介质，富集、筛选了芘的降解菌。 研究结果表明，石油污染土壤中的微生物群落结构主要与其相对地理位置有关，当污染物的浓度达到一定程度，土壤中的微生物群落结构会发生明显的改变。以13C标记的稳定同位素菲和芘为代谢底物，对污染土壤中菲和芘的土著降解菌群进行了鉴定。 13C-PLFA- GC-c-IRMS分析结果显示，参与菲降解的微生物有G-、G+细菌和放线菌，其中以G-细菌为主；污染土壤和相对清洁土壤两种土壤都未检出有真菌参与；参与芘降解的微生物有放线菌、G-细菌和真菌，其中以G-细菌为主，同时，也检测到有真菌的参与；污染土壤中的降解菌群较清洁土壤丰富。研究还发现，芘在土壤中的降解可能存在不经过菲为中间产物的代谢途径。采用不同的吸附性载体所富集的优势细菌显著不同。从不同的吸附性载体上最终得到8株芘的降解菌，其中鉴定出的三株菌中Sp-A56，Sp-A21为多食鞘氨醇杆菌（Sphingobacterium multivorum）；Sp-B05氨基杆菌属的一个亚种（Aminobacter sp.），未见有关该菌株芘降解能力的报道，可能是一株新的芘降解菌。

污水处理体系中的微生物群落特征研究

污水生物处理系统本质上是一种人工强化的工程化微生态系统。污水处理过程往往由多个功能互补的反应单元协同完成，例如因对污水中有机碳、氮和磷兼具良好的去除功能而在城市污水处理中被广泛应用的Anoxic / Oxic（A/O）生物处理工艺。不同反应单元特有的微生物群落之间的相互关系和相互作用与处理系统的稳定性和处理效率密切相关。所以对污水处理系统中微生物群落进行系统分析非常重要。研究系统中微生物群落的时空演替对于优化处理系统的设计和操作具有重要意义。但是，以往对于污水处理系统中微生态系统的解析多数针对实验室规模的其中个别反应器独立进行，还缺乏从系统水平对实际大规模运行的整个污水处理过程中所有反应单元群落进行分析的研究。 悬挂链移动曝气系统是对A / O工艺的完善和发展。悬挂链曝气工艺的实现是依靠悬挂链移动曝气设备和完善的自动控制系统来完成的。可以在系统中实现类似多级A/O的可能性，水力停留时间较长，污泥龄达到15天以上，能够完全实现A / O 工艺。 目前正被广泛应用在各种行业的污水处理项目中。 本文应用基于细菌16S rRNA中的PCR扩增方法（Polymerase Chain Reactor），结合变性梯度凝胶电泳指纹分离技术（Denaturing Gradient Gel Electrophoresis, DGGE），对实际规模的运用Anoxic / Oxic（A/O）工艺并采用悬挂链式移动曝气技术的污水生物处理系统中微生物群落特征，主要对细菌组成结构和群落动态，细菌优势菌群的多样性以及与系统功能稳定性的关系进行了研究，拟为更全面了解活性污泥处理系统中的优势菌群特征，以及细菌群落结构和功能动态变化关系，实现对活性污泥处理的优化操作，对污染物降解功能菌群的筛选，为运用现代培养技术实现分离培养并运用于环境修复实践奠定方法和理论基础。 首先，对影响PCR-DGGE分析的重要前操作步骤进行了优化和筛选，包括两个方面：细菌基因组DNA的高效提取和纯化；不同16S rRNA靶序列对PCR-DGGE分析的影响。从中选出适合于活性污泥样品的细菌基因组DNA提取方法和PCR-DGGE分析的最优靶序列组合。 其次，运用PCR-DGGE指纹图谱技术分析了该污水处理系统中不同功能反应单元中活性污泥的细菌种群结构特征，探讨了系统运行过程中细菌种群时间和空间上的动态特征。并将图谱中所显示的优势条带进行切割回收，重复扩增，电泳检测，序列测定并与GenaBank数据库中的微生物类群进行同源性比对，探讨活性污泥中细菌种群多样性，了解污泥中可能含有的主要具有污染物降解功能的类群信息。 在整个处理过程中，同一功能反应单元中不同位置的活性污泥微生物菌群结构不同。执行不同功能的处理单元活性污泥细菌多样性和组成结构各有不同。 在系统稳定运行的状态下，细菌组成结构的时间变化动态不显著。但是在系统的不同操作条件下，主要处理池的微生物群落的DGGE遗传指纹图谱较独特。 对该处理系统污泥中优势菌群的序列测定和同源性比对表明，优势菌群所对应的细菌的16S rDNA序列可以被归属于以下四个主要的细菌系：α, β, γ- Proteobacteria 以及厚壁菌门 phylum Firmicutes （low G+C Gram-positive）。 该处理系统的优势菌群的DGGE条带拥有潜在的具有异养硝化/好氧反硝化的除 N / P 类群。该类菌群中的大多数属于Pseudomonas spp.。另外，回收到两个与已鉴定的具有异养硝化和好氧反硝化能力的Pseudomonas stutzeri 和 Pseudomonas borbori 最相似的菌株的条带。γ-变形菌纲门（γ- Proteobacteria）的微生物类群在该缺氧-好氧处理厂中分布较广泛，尤其是和 N / P 去除紧密相关的具有脱氮除磷能力的Pseudomonas 类群，而且在好氧曝气处理池中分布较广，这可能和系统中表现的好氧反硝化现象相关。 不同的操作状况下微生物群落结构有差异。增加污泥回流比，增加DO（Dissolved oxygen）浓度，COD去除率和NH4+-N去除率显著增加，总N和总P的去除率改变不显著。 最后，对整个处理过程中微生物群落结构在系统正常调控改变范围内的长期动态和稳定性进行了探讨。整个处理系统的长期稳定性与体系中的每个处理环节相关，而不是仅与其中的单个主要反应池相关。污水处理体系的功能稳定性与其中的微生物群落稳定性相关，微生物群落结构决定了生态功能，群落结构变化能反应系统的运行状况及其降解效率。

青杨组(Populus Section Tacamahaca Spach)不同种对非生物胁迫的响应差异

土壤是人类赖以生存的自然环境和农业生产的重要资源，目前土壤受到干旱和盐胁迫的危害越来越严重。杨树具有适应性强、生长快和丰产等特性，本论文以青杨组杨树为模式植物，研究杨树对土壤干旱和盐胁迫的生态生理及蛋白质组学反应，研究成果可为我国干旱半干旱地区营造人工林、防止沙漠化提供理论依据，也为恢复与重建盐污染地区退化生态系统提供科学指导。主要研究结果如下： 1 青杨不同种对逐步干旱胁迫的响应差异 将来自喜马拉雅山东缘高海拔的康定杨和低海拔的青杨枝条扦插在温室中，用来检测它们对逐步干旱胁迫的响应。研究结果表明来自不同海拔的杨树对逐步干旱胁迫的适应性反应是不一样的。株高、叶片发育、叶片相对含水量、丙二醛、过氧化氢等指标的显著性变化在青杨中比在康定杨中来得早些，而且随着干旱胁迫程度的增加，这些参数的变化越来越明显，尤其是当青杨受到严重干旱胁迫的时候；而可溶性蛋白、可溶性糖、游离脯氨酸、抗氧化酶活力变化在康定杨中来得早一些。与青杨相比，在干旱胁迫下，康定杨仍能保持较好的植株生长和叶片发育；康定杨也能在逐步干旱条件下积累更多的可溶性蛋白、可溶性糖、游离脯氨酸及抗氧化酶活力，但是在丙二醛和过氧化氢含量方面增加的更少些。而且，我们的研究结果表明高海拔的康定杨有更强的耐干旱能力，杨树对干旱胁迫的适应能力与干旱发生的速度、强度、持续时间及两种杨树的海拔有关。 2 干旱胁迫下青杨不同种的蛋白质组学分析 来自青杨和康定杨雌株的枝条扦插在温室中，用来研究它们对干旱胁迫的蛋白质组学反应。采用TCA-丙酮/酚提取法提取总蛋白，并进行双向电泳分析。在每个处理的重复图像中都能检测到1,000 个以上的蛋白点。在青杨中有58 个蛋白在干旱处理后发生显著变化，其中22 个蛋白通过肽指纹图谱成功鉴定。康定杨中有69 个蛋白的表达量发生了显著变化，其中有25 个蛋白通过肽指纹图谱成功鉴定。这些被鉴定的蛋白主要参与了光合作用、氧化还原平衡、信号传导、能量代谢、蛋白质合成等过程。尽管被鉴定的蛋白只占叶片总蛋白的很少一部分，但这些被鉴定的干旱响应蛋白可能对维持植株内部平衡方面有重要作用。 3 青杨的盐胁迫响应 青杨植株分别用 0、50 和100 mM NaCl 溶液进行处理。叶片相对含水量、叶绿素a、b 含量、CO2 同化速率和气孔导度的降低表明叶绿体受到了盐胁迫的影响。过氧化氢、丙二醛含量及电导率的升高表明细胞受到了伤害。可溶性糖、游离脯氨酸含量及抗氧化酶含量的上升增加了植株耐盐胁迫的能力。在每个处理的重复图像中都能检测到1,000 个以上的蛋白点。其中有38 个盐响应蛋白被成功鉴定，有16 个蛋白(点4、10、11、14、15、21、24、26、27、28、33、34、35、36、37 和38)出现在盐胁迫的植株中；3 个蛋白(点10、11 和35)只出现在重度盐胁迫处理中；而1 个蛋白(点1)只出现在对照处理中。2 个蛋白(点1 和2)表达量下降，其余蛋白点表达量都增加。被鉴定的蛋白一部分参与了生理生化反应，而另一部分则在信号传导、蛋白质合成等方面有重要作用。盐胁迫下的生理生化变化及蛋白质组学的联合研究有利于青杨对盐胁迫的适应性分析。 Soil is the indispensable environment for human survival and important resource for agriculture development. Nowadays soil is threatened by drought stress and salt stress. Poplars (Populus spp.) possess some characters such as strong acclimilation, fast growth and great production of biomass. In this study, different species of Populus section Tacamahaca spach were used as model plants to investigate the ecophysiological and proteomic responses to drought stress and salt stress. Our results can provide theoretical evidence for the afforestation and prevention of desertification in the arid and semi-arid areas, and also can supply scientific direction for the reconstruction and rehalibitation of ecosystems contaminated by salinity. The results are as follows: 1 Adaptive responses to progressive drought stress in two contrasting poplar species originating from different altitudes Cuttings of Populus kangdingensis C. Wang et Tung and Populus cathayana Rehd., originating from high and low altitudes in the eastern Himalaya, respectively, were examined during one growing season in a greenhouse to determine the effects of progressive drought stress. The results manifested that the adaptive responses to progressive drought stress were different in these two species from different altitudes. Significant changes in height increment, leaf development, relative water content (RWC), malondialdehyde (MDA) and hydrogen peroxide (H2O2) appeared earlier in P. cathayana than in P. kangdingensis, whereas changes in soluble protein, soluble sugar, free proline and antioxidant enzymes appeared earlier in P. kangdingensis. In addition, changes in these parameters became more and more significant when the drought stress progressed, especially under severe drought stress in P. cathayana. Compared with P. cathayana, P. kangdingensis was able to maintain a superior height increase and leaf development under drought stress. Also, P. kangdingensis possessed greater increments in soluble protein, soluble sugar, free proline and antioxidant enzymes, but lower increments in MDA and H2O2 than did P. cathayana when the cuttings were exposed to progressive drought stress. Our results suggest that P. kangdingensis originating from the high altitude has a better drought tolerance than does P. cathayana originating from the low altitude. Furthermore, this study manifested that acclimation to drought stress are related the rapidity, severity, duration of the drought event and the altitude of two contrasting species. 2 Proteomic responses to drought stress in two contrasting poplar species originating from different altitudes The cuttings from a female clone of P. kangdingensis and P. cathayana were used to determine proteomic response to drought stress, respectively. Total proteins of the leaves were extracted by a combination of TCA-acetone and phenol, and separated by two-dimensional gel electrophoresis. More than 1,000 protein spots were reproducibly detected on each gel. 58 differentially expressed spots were detected under drought stress in P. cathayana and 22 drought-responsive proteins were identified by peptide mass fingerprint. 69 differentially expressed spots were detected under drought stress in P. kangdingensiss and 25 drought-responsive proteins were identified by peptide mass fingerprint. The identified proteins are involved in several processes, i.e., signal transduction, protein processing, redox homeostasis, CO2 fixation and energy metabolism. Although the proteins identified in this investigation represent only a very small part of the poplar leaf proteins, some of the novel drought-responsive proteins identified here may be involved in the establishment of homeostasis in response to drought stress in the woody plants. 3 Responses to salt stress in P. cathayana Cuttings from a female clone of P. cathayana were treated by Hoagland’s solution: 0, 50, 100 mM NaCl, respectively. Salinity significantly decreased the relative water content of leaves, the contents of chlorophyll a and chlorophyll b, CO2 assimilation rate (A) and stomatal conductance (gs) in both salt stress treatments，which suggested the chloroplast was affected by salt stress. The observed increases of H2O2 and malondialdehyde contents and electrolyte leakage suggested that salinity caused cellular damage, whereas the increases in compatible solutes and in the activities of antioxidant enzymes enhanced the salt tolerance. More than 1,000 protein spots were reproducibly detected on each gel, and 38 salt-responsive proteins were successfully identified by peptide mass fingerprint (PMF). 16 spots (spot 4, 10, 11, 14, 15, 21, 24, 26, 27, 28, 33, 34, 35, 36, 37 and 38) absent in the control sample were induced by the salt treatment, and three spots (spot 10，11 and 35) were present only in the severely salt-stressed treatment. The %vol of the differentially expressed proteins generally increased with progressing salt stress, except for the decreased %vol of two proteins (spot 1 and 2) under salt stress and the presence of spot 1 only in the control sample. Some of the novel salt-responsive proteins identified here may be involved in physiological, biochemical response to salt stress in P. cathayana, the other identified proteins play a role in numerous cellular functions, including signal transduction and protein processing. An integrated physiological, biochemical and proteomic approach was used here to systematically investigate salt acclimation in poplar.

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.

不同小麦品种（系）中主要矿质元素的含量比较及与品质的关系

采用微波消解、电感耦合高频等离子体原子发射光谱（ICP-AES）的方法,对62份不同小麦品种（系）中锌、铁、铜、钙、钠和钾的含量进行了测定。同时利用红外线品质测定仪对主要品质指标粗蛋白、湿面筋、沉降值进行了测定。结果表明，不同小麦品种（系）中各种矿质元素的含量存在差异，2006年小麦品种中铁含量变幅为18.55-58.19 ug/g，平均为30.83ug/g ，最高与最低的相差39.64ug/g；锌含量变幅为5.70-25.80 ug/g，平均为15.13ug/g ，最高与最低相差20.10ug/g。2008年小麦品种（系）中铁含量变幅为16.68-52.25 ug/g，平均为30.10ug/g，最高与最低相差35.58ug/g；锌含量变幅为12.29-33.47 ug/g，平均为21.11ug/g，最高与最低相差21.18ug/g；钙含量变幅为167.53-348.80ug/g，平均为248.59ug/g，最高与最低相差192.59ug/g；铜含量变幅为2.32-5.83 ug/g，平均为2.98ug/g，最高与最低的相差3.61ug/g；钾含量变幅为1822.71-4414.91 ug/g，平均为2617.87ug/g，最高与最低的相差2634.72ug/g；钠含量变幅为10.25-39.82 ug/g，平均为23.05ug/g，最高与最低的相差29.57ug/g。 两年不同小麦品种（系）中矿质元素的含量分析结果表明：铁、铜、钙、钠和钾含量年际变化不明显,说明小麦对铁、铜、钙、钠和钾的吸收较稳定；锌含量变化较大，可能受环境的影响比较大。分析各矿质元素含量与粗蛋白、湿面筋、沉降值及元素之间的相关关系，结果表明，锌含量与粗蛋白含量呈极显著正相关关系，相关系数为0.317，与湿面筋含量之间呈显著正相关，相关系数达到0.246；铁含量与粗蛋白含量呈显著的正相关关系，相关系数是0.262；铜、钙、钠和钾含量与粗蛋白含量、湿面筋和沉降值之间存在正相关，但不显著，其中钠与沉降值之间为负相关。表明施锌或铁对提高小麦粗蛋白和湿面筋有显著效应，其余矿质元素有促进作用但不明显。 利用RAPD分子标记技术对川育23、41058、川育20及其父母本进行分析，力图从分子水平找到小麦矿质元素含量之间的差异性，琼脂糖电泳结果表明不同的小麦品种（系）间扩增出了差异条带。 以上研究结果，将对筛选“微量营养强化型”小麦新材料，选育“微量营养强化型”小麦新品种奠定基础。 62 different wheat cultivars was digested with HNO3 in a tightly closed vessel heated under micro-wave,then contents of zinc，iron,copper，calcium，sodium and potassium were determined by inductively coupled plasma-atomic emission spectroscopy(ICP-AES)．The main indexes of wheat quality such as total protein、wet glu and sedimentation volume were detected by Infratec 1255 Food & Feed Analyzer at the same time.The obtained results showed that variation for all of the mineral elements concentrations among different cultivars were observed .In 2006, the amplitude variation of the iron content was 18.55-58.19 ug/g,the average value was 30.83ug/g,and 39.64ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the zinc content was 5.70-25.8 ug/g,the average value was 15.13ug/g,and 20.10ug/g between the highest-content cultivar and the lowest one.In 2008, the amplitude variation of the iron content was 16.68-52.25 ug/g,the average value was 30.10ug/g,and 35.58ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the zinc content was 12.29-33.47 ug/g,the average value was 21.11ug/g,and 21.18ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the calcium content was 167.53-348.80ug/g,the average value was 248.59ug/g,and 192.59ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the copper content was 2.32-5.83 ug/g,the average value was 2.98ug/g,and 3.61ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the potassium content was 1822.71-4414.91 ug/g,the average value was 2617.87ug/g,and 2634.72ug/g between the highest-content cultivar and the lowest one; the amplitude variation of the sodium content was 10.25-39.82 ug/g,the average value was 23.05ug/g,and 29.57ug/g between the highest-content cultivar and the lowest one. Analysis was made on the annual variation of mineral elements content in different Wheat cultivars ,the result shows:there is no obvious difference of iron ,copper ,sodium、calcium and potassium concentrations in wheat cultivars, suggesting the absorption of the iron, copper, sodium、calcium and potassium by wheat are relatively steady ,but zinc concentrations change obviously ,maybe influenced heavily by environment . The correlation between mineral elements 、mineral elements and total protein、mineral elements and sedimentation volume as well as mineral elements and wet glut were analysed in this paper, the result showed that there was significant positive correlation between zinc content and total protein (the correlation coefficient is 0.317), positive correlation between zinc content and wet glu (the correlation coefficient is 0.246), positive correlation between iron content and total protein (the correlation coefficient is 0.262). there was positive but not obvious correlation between the contents of copper, calcium, sodium or potassium and total protein, wet glut or sedimentation volume,among which was negative correlation between sodium and sedimentation volume.It was indicated zinc or iron fertilization has prominent effects in improving the total protein in wheat, the rest mineral elements have Non- obvious facilitation. The study then forecasted the genetic difference of different wheat by the molecular marker of RAPD in order to find differences in molecular level. Chuanyu23、41058、chuanyu20 as well as their male and female parents were analysed by RAPD markers,Agarose gel electrophoresis of DNA revealed the appearance of differential bands . The above-mentioned results of this study establish the foundation to screening the new materials of wheat of " strengthening type of micro- nutrition ", and to breeding the new wheat cultivars of" strengthening type of micro- nutrition ".

小麦胚乳蛋白组分和Glu-B3位点基因核心编码区差异与小麦品质关系的研究

小麦加工品质改良已成为我国小麦育种的主要目标之一。特别是我国加入WTO以后，对小麦产品的质量提出了更高的要求，小麦品质改良的任务将更加艰巨和重要，小麦胚乳蛋白是影响小麦加工品质性状的重要因素。因此，深入了解小麦胚乳蛋白对加工品质性状的影响及其分子基础，为品质改良提供理论依据和科学指导，对加速我国小麦品质育种和优质小麦生产具有重要意义。本研究选用在麦谷蛋白5个基因位点(Glu-A1、Glu-B1、Glu-D1、Glu-B3和Glu-D3)上均含不同等位基因的小麦品种99G45和京771及Pm97034和京771杂交F9代共164个麦谷蛋白纯合系，及228个中国推广普通小麦品种和高代育成品系为试材，研究了麦谷蛋白Glu-1和Glu-3位点基因等位变异对籽粒蛋白、湿面筋含量、Zeleny沉降值和SDS沉降值间的关系；本研究还利用小麦A、B和D基因组中低分子量麦谷蛋白亚基（LMW-GS）基因特异引物，通过PCR方法克隆了1个Glu-A3位点和3个Glu-B3位点LMW-GS基因片段，在此基础上分析了不同等位基因对品质造成差异的分子基础；另外，本研究对中国近年推广的部分品种和育成的高代品系资源的多样性进行了分析。现将主要研究结果简述如下： 1. 对来自三个麦区的148份材料的醇溶蛋白组成进行了分析，结果表明，各麦区醇溶蛋白模式具有较大差异。在ω区,A7、B、E、F、G、J、P、Q、S和U仅存在于西南秋播麦区；A3、M、N、R、W和X仅存在于黄淮特种麦区；K仅存在于北方冬麦区；A6是北方冬麦区出现频率最高的带型模式，而西南秋播麦区中D出现的频率最高。ω-区的E、H和M几种模式是以前国内外未曾报道的。且初步确定，这些模式对品质性状具有正效应。至于γ区,A、B、D、E和F在各区均有出现，其中B和E在各区出现的频率都很高，在26.1-39.6％之间。相反，H 仅出现在黄淮特种麦区，J仅限于西南秋播麦区。对于β-区醇溶蛋白，B型模式在所有区中都相当高，而模式A仅存在于第三区.对于α-区，模式A在Ⅲ区而模式D在Ⅱ区出现的频率很高。1BL.1RS易位系在中国小麦品种中出现频率高达41.2％，在I, II和Ⅲ麦区的出现频率分别为 45.5、43.5和35.2%。各生态区模式的差异可能是品种适应不同生态条件和人为选择的结果，但这有待进一步证明。由于醇溶蛋白位点（Gli-1）与LMW-GS位点（Glu-3）紧密连锁，本结果可为下面确定普通小麦LMW-GS等位基因变异所用。 2. 利用Gli-1与Glu-3的紧密连锁，以228个小麦品种/系为材料，首次对中国小麦品种麦谷蛋白亚基的6个位点进行综合分析，研究小麦籽粒蛋白与品质性状间的关系，结果表明6个高分子量（HMW）和低分子量（LMW）麦谷蛋白位点对蛋白质含量的效应大小为，Glu-D1>Glu-B3>Glu-A1=Glu-B1> Glu-A3=Glu-D3；对GMP含量的效应大小为, Glu-A3>Glu-B3>Glu-D1> Glu-B1>Glu-A1>Glu-D3；对湿面筋含量的效应大小为, Glu-B1>Glu-B3= Glu-D3>Glu-A3>Glu-A1>Glu-D1；对Zeleny沉降值的效应大小为, Glu-A1> Glu-B3>Glu-D3>Glu-D1>Glu-B1>Glu-A3；对SDS沉降值的效应大小为, Glu-B3>Glu-A1=Glu-D1=Glu-A3>Glu-D3>Glu-B1。对蛋白含量而言，各位点的最佳组合方式为1、17+18、5+10、Glu-A3e、Glu-B3g、Glu-D3b；对湿面筋含量而言，各位点的最佳组合方式为1、6+8、5+10、Glu-A3d、Glu-B3c、Glu-D3b；对Zeleny沉降值而言，各位点的最佳组合方式为N、17+18、5+10、Glu-A3d、Glu-B3d、Glu-D3b；对SDS沉降值而言，各位点的最佳组合方式为1、7+8、2.2+12、Glu-A3b、Glu-B3g、Glu-D3b。另外，分析了稀有亚基对5+12与2.2+12与品质性状的关系，认为5+12对品质有负效应，2.2+12对品质有正效应。在品质育种时，应对优异组合或优异亚基加以利用。 3. 首次利用重组自交系（RILs）为材料，研究麦谷蛋白亚基表达量与品质性状的关系，通过对重组自交系中各HMW-GS表达量的分析，认为，就单个亚基的表达量而言，7亚基最高；其次为2亚基、5亚基、12亚基和10亚基；亚基9和1的表达量最小；N亚基不表达。对成对出现的亚基对而言，x型和y型亚基的总表达量2+12>5+10>7+9>17+18。就单个亚基与品质性状的关系而言，仅有10亚基的表达量与蛋白含量的相关性达5%的显著水平，2亚基的表达量与湿面筋含量呈负相关，显著水平也达5%，其余单个亚基对品质性状均无显著影响；就x型/y型亚基的比例来看，2/12和5/10对湿面筋含量都有显著的负效应；对某一位点等位基因控制的亚基表达总量来看，2+12对SDS沉降值有显著负效应。另外，本研究得出：2＋12的亚基对的负效应主要体现在2亚基上，且在同一位点上，x型亚基的表达量大于y型。所以推导稀有亚基组合2+10很可能也是劣质亚基。 4. 以 Glu-A1、Glu-B1、Glu-D1、Glu-B3和Glu-D3作为5个因素对99G45/京771和Pm97034/京771杂交后代的蛋白质含量和SDS沉降值进行多因素方差分析。结果表明，Glu-A1和Glu-D3对蛋白含量的加性效应达5％显著水平；Glu-D1 * Glu-D3对蛋白质含量的互作效应也达5%显著水平；其余位点的加性和互作效应对蛋白质含量的影响均不显著。对SDS 沉降值而言，Glu-D1的加性效应最大,贡献率为4.2 % ,达1 %显著水平,其次是Glu-B1位点,贡献率为3.3% ,达5%显著水平。其余位点对SDS 沉降值的加性和互作效应均未达5%显著水平。总体而言, 各位点对蛋白含量的效应大小为Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3；对SDS沉降值的效应大小为Glu-D1>Glu-B1> Glu-D3>Glu-A1> Glu-B3。Glu-D1和Glu-D3位点上等位基因变异对蛋白含量有显著或极显著影响，含Glu-D1d和Glu-D3 GD、Glu-D3 JD基因的株系分别比含Glu-D1a和Glu-D3 PD基因的株系有较高的蛋白含量；在该遗传背景下，麦谷蛋白各基因位点对蛋白含量的效应大小依次排列为：Glu-A1位点1>N；Glu-B1位点7+9>17+18>14+15；Glu-D1位点5+10>2+12；Glu-B3位点GB>JB>PB；Glu-D3位点GB>JB>PB。对SDS沉降值的效应大小依次排列为：Glu-A1位点1>N；Glu-B1位点7+9=17+18>14+15；Glu-D1位点5+10>2+12；Glu-B3位点GB>JB>PB；Glu-D3位点GB>JB>PB。所以，对蛋白含量和SDS沉降值均较好的组合为1，7+9，5+10，GB，GD。 5. 因为GB和PB对品质的效应有显著差异，选取LMW-GS位点特异扩增引物对京771、99G45和Pm97034的Glu-B3位点进行扩增，结果得到三个不一样的扩增片段（Genebank号为DQ539657－DQ539659），得到的基因片段与Genebank中已报道的同类序列高度同源。通过克隆片段组成的分析，发现对Pm97034的序列较京771和99G45段少一个7氨基酸的重复单元，这可能是它较另外两个片段对面筋强度影响小的主要原因；另外，在99G45的序列中，124位处出现L（亮氨酸）代替P（脯氨酸），158位处出现了T（苏氨酸）代换M(蛋氨酸)，这可能是99G45Glu-B3位点序列对SDS沉降值的效应显著优于Pm97034的原因。 6．通过对RILs各位点同普通小麦品种（系）各位点与品质关系的比较，发现对SDS沉降值的效应，各位点在不同研究材料中是不同的，普通小麦中：Glu-B3>Glu-A1=Glu-D1=Glu-A3>Glu-D3>Glu-B1，RILs中：Glu-D1>Glu-B1> Glu-D3>Glu-A1> Glu-B3。利用重组自交系材料（完全排除了1BL/1RS易位干扰）所得到的结果与Gupta and MacRitchie (1994)所得结论一致。进一步证实了1BL/1RS易位对小麦品质的重要影响。对蛋白含量而言，普通小麦品种（系）中，Glu-D1>Glu-B3>Glu-A1=Glu-B1> Glu-A3=Glu-D3，RILs中，Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3，和对SDS沉降值的效应一样，推断在非1BL/1RS易位的情况下，各位点对其效应应为Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3。 对同一位点的等位基因而言，普通小麦和重组自交系中Glu-A1和Glu-D1上的等位基因对品质性状的贡献是一致的，但Glu-B1上的等位基因对SDS沉降值的贡献发生了变化，普通小麦中17+18>7+9，RILs中7+9>17+18，这可能也是1BL/1RS造成的。 Baking quality improved is one of the main object of wheat bread in China. The overall objective of the present studies was to increase the understanding about protein quality in wheat, i.e. to make it possible to improve the production of wheat with desired quality for different end-uses. With the analysis of gluten protein in RILs, 99G45/Jing 771 and Pm97034/Jing, and 228 wheat cultivars or lines in China, the correlations between glutenin compositions and protein content, glutenin macropolymer(GMP), wet gluten content, Zeleny sedimentation value and SDS sedimentation value contentand breadmaking quality were studied. Also a rapid and efficient detection method of geneticpolymorphism at Glu-B3 loci in wheat was established using polymerase chain reaction(PCR).The results obtained were as follows: 1. Cultivated Chinese wheat germplasm has been a valuable genetic resource in international plant breeding. Patterns of gliadin among cultivated Chinese accessions are unknown, despite the proven value and potential novelty. The objective of this work was to analyse the diversity within improved Chinese wheat germplasm. The electrophoretic banding patterns of gliadin in common wheat cultivars and advanced lines were determined by acid-polyacrylamide gel electrophoresis. For 148 leading commercial cultivars and promising advanced lines used in our study, 48 patterns were identified, 29 corresponding to ω-gliadin, 9 to γ-gliadin, 5 to β-gliadin and 5 to α-gliadin. The most frequent patterns were A6 in ω; B in γ; B in β and A in the region of α. 116 band types appeared in the148 samples: 94 accessions had unique gliadin types, and 22 gliadin types while not unique were found in 54 accessions. The gliadin patterns of Chinese wheat cultivars and lines greatly differed from the patterns of wheat lines from other countries. Three patterns, E, J, H, M, N and O in the ω-zone had not previously been reported. Three wheat zones，the Northern Winter Wheat Region, the Yellow and Huai Valley River valleys Winter Wheat Region and the Southwestern Winter Wheat Region，in China showed different frequencies in their gliadin patterns. This information can be used to monitor genetic diversity with Chinese wheat germplasm. 2. To analyse the relationship between the loci and characteristics quality, we utilized the 228 cultivars/lines. The results showed that : For protein content, Glu-D1 >Glu-B3>Glu-A1=Glu-B1>Glu-A3=Glu-D3. For GMP content, Glu-A3>Glu-B3 >Glu-D1>Glu-B1>Glu-A1>Glu-D3. For wet gluten content, Glu-B1>Glu-B3= Glu-D3>Glu-A3>Glu-A1>Glu-D1. For Zeleny sedimentation value, Glu-A1>Glu-B3> Glu-D3>Glu-D1>Glu-B1>Glu-A3, For SDS sedimentation value, Glu-B3>Glu-A1= Glu-D1= lu-A3>Glu-D3>Glu-B1。For protein content, the best combination of 6 loci is (1,17+18,5+10,Glu-A3e, Glu-B3g,Glu-D3b). For wet gluten content, the best combination of 6 loci is (1,6+8,5+10,Glu-A3d,Glu-B3c,Glu-D3b). For Zeleny sedimentation value, the best combination of 6 loci is (N,17+18,5+10,Glu-A3d, Glu-B3d, Glu-D3b). For SDS sedimentation value, the best combination of 6 loci is(7+8,2.2+12,Glu-A3b, Glu-B3g,Glu-D3b)。Additional, we analysed the relationship between the subunits 5+12 and 2.2+12, think that 5+12 was negative for quality, 2.2+12 is postive for quality. It should be effective utilized. 3. It’s the first time to utilize RILs to study the relationship between subunits expression quantity and characteristics quality. The results showed that: For single subunit, the expression quantity of 7 is the highest. Then the 2, 5, 12 and 10. The expression of subunit 9 and 1 is the lowest. Subunit N is not expressed. For subunits, the expression quantity of x type and y type are 2+12>5+10>7+9>17+18. The significant relation of 5% only showed between the expression quantity of subunit 10 and protein content. The relationship between expression quantity of others and characteristic quality was not significant. For x type/ytype, 2/12 and 5/10 is negative relation insignificant level. For the subunit(s) in a loci, Only 2+12 effect SDS sedimentation value negative in significant level. 4. With RILs 99G45/Jing 771 and Pm97034/Jing 771, we found that: The effective of Glu-A1, Glu-D3 and Glu-D1 * Glu-D3 for protein content is significant at 5% level. The effect of other loci for protein wre not significant. For SDS sedimentation value, the effect of Glu-D1is the highest, which contribution is 4.2 % .Then the Glu-B1, contribution is 3.3%. The effect of other loci for SDS sedimentationvalue were not significant. In total, for protein content: Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3; for SDS sedimentationvalue: Glu-D1>Glu-B1> Glu-D3>Glu-A1>Glu-B3. The effect of alleles in Glu-D1 and Glu-D3 loci are significant at 1% or 5%. In Glu-A1, 1>N; Glu-B1, 7+9>17+18>14+15; Glu-D, 5+10>2+12; Glu-B3, GB>JB>PB; Glu-D3, GB>JB>PB. For SDS sedimentation, Glu-A1, 1>N; Glu-B1, 7+9=17+18>14+15; Glu-D1, 5+10>2+12; Glu-B3, GB>JB>PB; Glu-D3, GB>JB>PB. The best combinations for SDS sedimentation value is 1，7+9，5+10，GB，GD. 5. Because of the difference of GB and PB for SDS sedimentation value, we selected the specific primer for LMW-GS loci to amplified the Glu-B3 of Jing771, 99G45and Pm97034. We got 3 amplify fragment (Gene Bank accession number are DQ539657－DQ539659）. We found that the fragment of Pm97034 were deleted a repetitive 7 amino acid domain, which is perhaps the reason effect the gluten strength. Furthermore, in the position 124 of sequence 99G45, L has been replaced with P. Position 158, T replaced M, which may be the reason why the Glu-B3 locus of 99G45 is prefer to Pm97034 when refer to SDS sedimentation value. 6. Comparing the results of RILs and common wheat, we found that perhaps just the1BL/1RS made the difference of loci in different accession.

溶藻菌群对铜绿微囊藻的溶藻效应及其作用机理研究

水华暴发是一个世界性的问题，近年来在发展中国家显得尤其严重。水华暴发给环境和公众健康带来巨大灾难，一些蓝藻产生的毒素可以造成鱼类、鸟禽和家畜的死亡，而臭名昭著的微囊藻产生的微囊藻毒素更是有强烈致癌效应。因此，寻找控制水华藻类的有效方法非常迫切。在利用物理和化学方法处理不甚理想的情况下，利用溶藻细菌控藻成为一个新的研究方向。溶藻细菌一般直接从富营养化水体中分离，杀藻活力对有害蓝藻具有较强的选择性而不危害其它生物，尤其适合在水华发生初期使用，可以在短时间内达到阻止藻类增殖的效果。本研究富集分离到一个高效溶解铜绿微囊藻的溶藻菌群，对其溶藻效应和溶藻机制进行了探索研究。 1溶藻菌群的富集筛选及其溶微囊藻效果 富集筛选得到一个有明显抑藻效果的菌群，它对铜绿微囊藻有显著溶藻效果。与对照组相比，加入富集的溶藻菌后，第4 d开始出现溶藻现象，6~8 d出现明显的溶藻效果，8 d后测得叶绿素去除率在85%以上。 2 溶藻菌群的作用范围及溶藻特性 富集分离到的溶藻菌群对铜绿微囊藻和念珠藻有显著溶藻作用，对水华微囊藻和其它几株受试微囊藻没有明显溶藻效应。该溶藻菌群不仅可以在液体中溶解铜绿微囊藻，生长在固体平板上的藻苔也有一定的溶藻效应，生成溶藻空斑。保证快速溶藻的最大稀释度可以达到1/100, 000。 3 环境因子对菌群溶藻效力的影响 试验发现，不同的pH、温度、和光照条件下，溶藻菌群溶藻效力明显不同，且不同种类的氮源对其溶藻作用也有一定影响。这些条件对该菌群溶藻作用的影响，在相当的程度上可能取决于它们对藻和细菌两者的生长状况的影响综合。 4 溶藻菌群的溶藻作用机理 溶藻菌液过滤除菌和煮沸灭菌处理后溶藻液，未见明显的溶藻效果，只有原液具有很好的溶藻效果。因此可初步确定，蓝藻细胞的溶解可能是由溶藻菌直接接触藻细胞产生的作用效果。显微镜观察发现，细菌在溶藻的过程中频繁地接触藻细胞并侵入藻细胞，破坏进而裂解杀死藻细胞。这也进一步说明了此溶藻菌是通过直接方式杀藻。 5 溶藻菌群的菌群结构解析 分离有溶藻效果的纯菌的多次尝试都没有成功。结合DGGE和16S rDNA文库综合分析发现：Rubritepida菌，假单胞菌和鞘氨醇单胞菌是存在于铜绿微囊藻中的三种伴生细菌。加入富集的溶藻菌群后，菌群结构发生明显的变化，Rubritepida菌、假单胞菌消失，混合菌群则包含未培养黄杆菌，鞘氨醇单胞菌和噬氢菌，其中黄杆菌是优势菌群，并且细菌种群结构的变化与藻细胞消亡之间有显著的相关性。通过菌种的分离鉴定与DGGE和16S rDNA文库的测序结果比较，一些未培养菌可能在溶藻过程中起重要调控作用。 6 溶藻细菌控藻应用基础 (1) 扩大规模的模拟水华实验进一步确定了细菌对微囊藻的强烈溶解作用。 (2) 铜绿微囊藻(Microcystis aeruginosa 905, zc)、微囊藻(Microcystis spp., zd)和溶藻菌群共培养试验表明，zc可以抑制zd生长，而溶藻菌群可以溶zc。 本研究是第一次报道混合菌群的溶藻效应。该溶藻菌群对带有藻际细菌的铜绿微囊藻具有高效的溶藻效力，表明它对自然界中存在的带菌铜绿微囊藻和其它一些蓝藻的生消具有一定的控制作用。对进一步研究菌藻关系与生态学作用，以及对富营养化湖泊和水库水体中蓝藻暴发的防控，该菌群具有一定的应用潜力。 Cyanobacterial blooms break out frequently all over the world, especially in developing countries. Blooms create enormous disasters to public health and to the environment. Some cyanobacterial blooms produce extremely toxic substances that have killed fish, domestic animals and birds. It has been well known that microcystins, a hepatoxin produced by Microcystis, can promote tumors in humans. So it is very important to find an effective method for controlling the growth of the bloom-forming algae. Measures for controlling such kind of algae include physical, chemic and biologic means, but the former two may damage the aquatic environment and require high-energy inputs. The alternative approach for the elimination of nuisance algae involves the application of algicidal bacteria. The algicidal bacteria, which are nontoxic to other organisms and most of which are isolated from the eutrophic lake in situ, may be potential microbial algaecides. In the initial stages of the water blooms, they are able to restrain the biomass or multiplication of the bloom-forming algae in a short time. In order to use algicidal bacteria to suppress blooms of M. aeruginosa, we isolated a bacterial culture capable of lysing the noxious cyanobacteria M. aeruginosa. In this paper we described some properties of the bacterial culture and its growth-inhibiting or algicidal effects on the growth of M. aeruginosa, and investigated its algicidal mechanisms. 1 Enrichment of a microbial culture that lyses Microcystis aeruginosa A mixed bacterial culture was isolated from a hypereutrophic pond and showed significant algicidal activity against the noxious Microcystis aeruginosa. Algae lysis would be seen obviously 4 days later when the algae culture was killed and became yellow contrast to no-addition controls, and chlorophyll a (chl-a) reduction went beyond 85% 8 days later. 2 The host range and some other algicidal feature of the mixed algicidal culture. Microcystis aeruginosa, Nostoc sp., were susceptible to the mixed algicidal culture, while the lytic effects of this mixed culture on Microcystis flos-aquae and some other tested Microcystis were feeble.The algicidal culture can not only lyse M. aeruginosa in liquid media, but aslo lyse M. aeruginosa lawns on soft agar plates and form plaques. The maximun dilution of the mixed culture required for rapid Microcystis lysis is 1/100, 000. 3 Influences of environmental factors such as pH, temperature, illumination, and the nitrogen source on the lytic activity of the mixed bacterial culture on Microcystis aeruginosa. In our investigations, it was shown that the lytic activity of the mixed bacterial culture on Microcystis aeruginosa was straightly correlated with pH, temperature, illumination, as well as the nitrogen source in the medium. The impacts of these environmental factors on the algicidal activity of the mixed bacterial culture, to a certain extent, may depend on both the algal and the bacterial growth rates under the tested environmental conditions. 4 The mechanisms of algal cell lysis by the algicidal bacteria Death was detected when the mixed bacterial culture was added to the algal culture, but not when only the culture filtrate or autoclaved bacterial culture was added. This indicates that the mixed bacterial culture did not release extracellular products inhibitory to Microcystis aeruginosa. In addition, under the microscope, we observed frequent contacts btween bacteria and algae cells, and some bacteria can even penetrate into target algal cells and destroyed them. These results may suggest that the bacterium kill the alga by direct contact. 5 Molecular Characterization of the algicidal bacterial culture Attempts for isolation of pure bacterium or bacteria from the enrichment culture responsible for Microcystis lysis have so far been failed. Based on PCR-DGGE (denaturing gradient gel electrophoresis) and 16S rDNA clone library analysis, Rubritepida sp., Pseudomonas sp. and Sphingomonas sp., as accompanying bacteria, were existed in M. aeruginosa. The bacterial community in M. aeruginosa showed significant change after adding the enrichment culture, where uncultured Flavorbacterium sp., Sphingomonas sp. and Hydrogenophaga sp. were observed, and the uncultured Flavorbacterium sp. became a dominant species. The obvious correlation can be seen between change of bacterial population and extinction of M. aeruginosa. Compared identification of pure bacterium with sequencing of DGGE bands and the clone distribution of the clone libraries, it was inferred that some uncultured bacteria were probably play an important role in controlling the growth and abundance of M. aeruginosa. This report is the first example of a mixed bacterial culture with the ability to lyse M. aeruginosa. 6 Further study for algae control by applications of algicidal bacteria (1) Algae lysis would be seen obviously 6 days later when the algae culture was killed and became yellow contrast to no-addition controls, and chlorophyll a (chl-a) was reducted to a low level 20 days later in the simulated water bloom experiments. (2) The growth of Microcystis sp. (zd) was restrained by Microcystis aeruginosa 905 (zc) when they were co-cultured together, and zc was lysed by the algicidal bacterial culture. This report is the first example of a mixed bacterial culture with the ability to lyse M. aeruginosa, and its algicidal activity remained high against non-axenic tested M. aeruginosa, suggesting that bacteria in the natural environment could play a role in controlling the growth and abundance of M. aeruginosa and other cyanobacteria. Such bacteria could also potentially be used as agents to prevent the mass development of cyanobacteria in eutrophic lakes and reservoirs.

自养氨氧化混合种群特性和共存机理初步研究

组特殊自养氨氧化混合种群，表现：无机环境种群生长迅速、生物量高；在一个完全无机的自养生长环境中，不仅保持高氨氧化速率，并出现丰富的异养微生物种群；该种群置于异养、厌氧环境中，迅速表现出产氢特征。对于这样一个特殊的生态体系，研究其共生机理，以及联接这些种群之间的碳源和能源问题，将具有非常重要意义。我们拟从种群特征、细胞表面分泌产物、游离体系产物多糖、蛋白和脂肪酸方面开展研究。 第一部分，自养氨氧化混合种群的基本特征。采用氨氧化培养基，进行种群氨氧化特征研究；采用扫描电镜观察自养混合种群的微观特征；沉降、离心去除微生物种群，分析水相中的总有机碳、糖类等物质；利用LB培养基进行种群的分离、纯化，并采用DGGE手段对微生物种群结构进行分析。结果表明，接入菌种后（2/5000（V/V）），培养液中氨（200mg/L）在3－5天内快速降解；亚硝酸盐与氨氮变化呈负相关趋势，仅有少量硝酸盐含量（< 30mg/L）。氨氧化种群的生物量增长与氨氧化趋势一致，初始生物量7.75 mg/L(蛋白含量)，3-5天后生物量快速增长，并达到最高63.06 mg/L（蛋白含量）。电镜图片显示，种群外包裹一层粘液。离心除去菌体后，检测培养液总有机碳和糖的含量，同样表现出与生物量增长相似的特征，分别由初始的3.73、2.35 mg/L，3－5内天迅速增加，并分别达到最大值35.19、27.45 mg/L。经初步分离、纯化并对纯化菌株进行测序，获得了10株异养微生物分别为布鲁氏菌科苍白杆菌属、纤维单孢菌、类芽孢菌属、黄杆菌属、无色杆菌、鞘脂单胞菌、嗜麦芽寡养单胞菌、噬氢菌属、硫红球菌、假单胞菌；DGGE显示，约有20分条离带，我们对其中的两条优势条带进行切割回收测序，鉴定为欧洲亚硝化单胞菌（Nitrosomonas eur）。 第二部分：混合种群自养-异养菌共生的可能机制。在对微生物种群特征初步分析基础上，针对胞外糖类组分可能被微生物代谢分解，我们重点对微生物细胞蛋白质与糖类进行分析。采用超声结合RIPA裂解液裂解，SDS-PAGE电泳分析混合种群总蛋白种类，并通过氨基酸分析仪及红外光谱法分析氨基酸组成及蛋白红外特征。采用超声破碎结合反复冻融对细胞样品进行处理，提取液采用醇沉、Sevage脱氮白，凝胶过滤方法脱盐和分级分离。对提取物的糖分析包括：紫外扫描，红外光谱，核磁共振，单糖组成分析；扫描电镜观察菌群破裂现象。SDS-PAGE分析结果表明：氨氧化种群不同生长阶段都显示出42kD蛋白表达量很高，d4时42kD蛋白表达已经很强，4-7d内一直持续这种过量表达，直到d8后表达开始减弱。说明42kD蛋白可能与氨氧化密切相关。红外光谱分析显示：细胞提取物的特征峰分布在3427.42cm-1、1718.18 cm-1和1681.72 cm-1、1160.07和1086.74 cm-1，分别对应为OH、 C=O、C－O－C基团，表明具有蛋白的典型特征；氨基酸分析显示蛋白中的Gly,Asp,Ala,Glu含量相对较高。 提取物中胞外多糖分离谱图得到不均一组分，共得到6个收集峰；紫外扫描在201－213 nm处有多糖吸收峰，同样表明多糖成分不均一性；多糖红外光谱特征峰主要分别在3400.49 cm-1、2920.28 cm-1、1154.54和1087.52 cm-1，对应OH、－CH2－ or CH 、C－O－H or C－O－C等多糖特征基团；多糖提取物核磁共振1H d4.3～5.9之间出现强吸收峰，这是1H中，多糖存在的明显证据，1H NMR中，其中O-乙酰基的甲基上的氢信号为d1.1～1.3之间。糖肟全苯甲酸酯衍生物的HPLC测定中，得到单一的单糖峰，由于时间问题，还未进行更深入的试验；电镜图片显示，种群中的细胞有大量的破裂现象。 实验表明，自养氨氧化混合种群显示出快速的氨氧化速率，氨氧化过程生物量和有机质的增加明显。微生物种群包裹粘液层，并分离纯化出大量的异养菌；去除菌体后的游离培养液中存在有机质（包括多糖）说明无机自养生长体系中存在异养菌生长、繁殖的二次碳源；细胞提取物中蛋白条带数目多、种类丰富；细胞多糖提取物具有明显的多糖特征，以及单糖的存在。结合种群的显微特征和游离体系中的有机质的检测结果，我们认为，无机自养生长体系中，种群细胞生长过程中发生的破裂现象可能是导致大量的蛋白、多糖释放到游离胞外，并成为其他异养菌生长的碳源和氮源。这可能是自养体系中，大量异养菌共生的可能机制，至于是什么原因引起种群生长过程中产生的破裂现象，还有待下一步深入研究。 A group of mixed autotrophic ammonia oxidizing populations, having much biological characteristic tested by concerned personnel for pilot test: Performed rapid population growth and obtained high biomass in inorganic environment; Not only maintained a high rate of ammoxidation, promoted a wealth of heterotrophic microbial populations growth in a totally inorganic and autotrophic growth environment; Placed in heterotrophic and anaerobic environment,had the performance characteristics that could rapidly produce hydrogen.For such a special ecological system, Study its symbiotic mechanism and the connection between these populations of carbon and energy issues, will have a very important significance. We intended from the characteristics of the population, the secretion product of cell surface, free substance in the liquid medium like polysaccharide, protein and fatty acids carrying out research. Part I: The basic features of mixed autotrophic ammonia oxidizing populations . Use inorganic liquid medium, processed study for ammonia oxidation characteristics of the population; we used scanning electron microscopy to get micro-features of autotrophic ammonia oxidizing populations .The medium was carried out settlement and centrifugal then removed the microbial populations, after all of that we analysis the water phase for total organic carbon(TOC), carbohydrate and other substances; Solid ammonia oxidizing medium was adopted to separation and purification of population, DGGE means was for structure analysis of microbial population. The results showed that after the inoculum of bacteria (2 / 5000 (V / V)), ammonia in the culture medium (200 mg / L) was rapid degradation in 3-5 days; ammonia and nitrite have the negative correlation between changes in the trend, then only a small amount of nitrate content (<30mg / L). The biomass growth of ammoxidation population in line with the trend of ammonia oxidation, the initial volume of it was 7.75 mg / L (protein content), in 3-5 days upto 63.06 mg / L (protein content). Electron microscope image showed, the populations were wrapped in a layer of mucus, including the a large number ruptted micorbe , Centrifuge to remove bacteria, then detected the medium for total organic carbon and sugar content, result took on the same characteristics with biomass growth, that were from the initial 3.73、2.35 mg / L respectively, in 3-6 days achieved rapid increase in the maximum to 35.19、27.45 mg / L respectively. After initial separation、 purification ,then processed sequencing to strains purified and got the result that there were 10 heterotrophic microorganisms : Brucella Branch pale bacillus, Cellu lomonas, Bacillus species category, a Flavobacterium, colorless Bacteria, Aeromonas sheath fat, little support maltophilia Aeromonas, macrophages species hydrogen, sulphur-MI, Pseudomonas bacteria spores; DGGE display, there were 20 separation bands approximately. Part II: Mixed populations that autotrophic - heterotrophic bacteria symbiotic mechanism. On the basis of preliminary analysis of microbial population characteristics, aiming at extracellular carbohydrate components might be decomposition by microbial, we focused on microbial cell protein and carbohydrate analysis. Using ultrasound combined with RIPA lysis cracking the cells, SDS-PAGE electrophoresis analysis the total protein species of the population, and through the amino acid analyzer studied the compositions of amino acid and infrared spectroscopy analysis of a protein infrared characteristics. Using ultrasound combined with repeatedly freezing and thawing to treated the cell sample, then took the means that alcohol precipitation, deproteinization by Sevage, gel filtration aimed at desalination and grade separation to deal with the lysates . The extraction of sugar analysis included: UV scanning, IR, NMR, single-sugar composition analysis. SDS-PAGE analysis showed that: 42 kD protein expression was very high at different growth stages of mixed autotrophic ammonia oxidizing populations , on the fourth day, 42 kD protein expression had been very strong, 4-7d, it had continued this excessive expression, then started to weaken after 7 days. 42 kD protein that might be closely associated with ammonia oxidation. Infrared spectral analysis showed that: cell extracts with the characteristic that the peak distribution in 3427.42 cm-1、1718.18 cm-1 and 1681.72 cm-1、1160.07 cm-1 and 1086.74 cm-1 corresponding to OH、C = O、C-O-C Groups which had the typical characteristics of protein; and analysis showed that amino acids including Gly, Asp, Ala, Glu ,the content in the protein is relatively high. Exopolysaccharide in the extracts had the separation map that it was uneven, received a total of six collection peaks by the detection mode of phenol-sulphruic acid method ; ultraviolet scan in the 201-213 nm department had polysaccharide absorbing peak, the same ingredients that polysaccharide heterogeneity; infrared polysaccharide spectral characteristics of the main peak at 3400.49 cm-1, 2920.28 cm-1, 1154.54 and 1087.52 cm-1, corresponding OH,-CH2-or CH, C-O-H or C-O-C;and other characteristics of polysaccharide group; 1H NMR of polysaccharide extract appeared absorption peak between d4.3 ~5.9, which is the apparent evidence of polysaccharide, In 1H NMR, the hydrogen signal of one of O-acetyl was between 1.1 to 1.3. The determination of Sugar oxime whole benzoate derivatives by HPLC, there was a single-sugar peak, as a matter of time, yet more in-depth test. Summary: Mixed autotrophic ammonia oxidizing populations show us that it had the ability in ammonia oxidizing and it was great, organic matter and biomass increased significantly in the process of ammonia oxidation. Microbial populations was wrapped up slime layer, the phenomenon of cell breakdown obviously, and there were a lot of separation and purification of the heterotrophic bacteria; a lot of organic matter (including polysaccharides)remined in the medium that removal of cell indicated the inorganic system existed secondary carbon sources that could be used by the heterotrophic bacteria ; there were a large number proteins bands of cell extract, rich variety; cell extracts of polysaccharide had obvious characteristics of polysaccharide, and the existence evidence of single-sugar. Combined population of microscopic characteristics and free of organic matter in the test results, we believe that the health of inorganic system, population growth occurred in the course of the breakdown of the phenomenon is likely to lead to a lot of protein and polysaccharide released into the extracellular free, And other heterotrophic bacteria use them to the growth as carbon and nitrogen. This may be autotrophic system, the large number of heterotrophic bacteria symbiotic mechanism.