活性污泥宏基因组芯片DNA的制备方法

宏基因组芯片的探针是通过构建环境DNA的宏基因组文库而获得的,为了获得理想的活性污泥宏基因组文库和宏基因组芯片探针,必须获得高质量的活性污泥DNA.本文作者采用8种不同的DNA提取方法提取活性污泥的总DNA,并通过比较DNA总量、纯度、完整性、是否含有PCR酶抑制剂、能否进行限制性内切酶酶切等指标来评价不同提取方法对活性污泥总DNA提取效果的影响.结果表明,溶菌酶-SDS-蛋白酶K-酚氯仿抽提处理活性污泥所提取的总DNA效果最好,提取的DNA总量多、纯度高,DNA片段大于23 kb,无需进一步纯化就可直接进行PCR扩增反应或SauA Ⅰ限制性内切酶酶切,但DNA如果需要EcoRⅠ、HindⅢ等限制性内切酶酶切,则需采用Roche公司琼脂糖凝胶回收试剂盒将DNA进一步回收纯化.图7表3参14     

深海沉积物宏基因组文库中产蛋白酶克隆的筛选及性质分析

提取东太平洋深海沉积物宏基因组DNA,构建了未培养微生物宏基因组文库.该文库平均插入片段30 kb以上,含有7 000个克隆,含外源DNA总长度约为210 Mb.从文库中筛选到18个产胞外蛋白酶的克隆,16S rRNA分析表明,它们分别来源于假单胞菌(Pseudomonas)、寡养单胞菌(Stenotrophomonas)和弧菌(Vibrio)3个菌属.根据活性大小及来源菌株的16S rRNA序列比较,选择10个蛋白酶进行酶学性质分析,结果表明,它们的最适作用pH值在7～10之间,最适作用温度在10～40℃之间.其中Pro20蛋白酶最适作用温度最低,为10℃;Pro1蛋白酶具有最适作用温度低(20℃)、pH耐受性好、热稳定性较好等特性,具有良好的应用开发潜力.图6参16     

基于宏基因组技术获得的对厌氧氨氧化菌代谢的新理解

厌氧氨氧化菌(Anaerobic ammonium oxidation bacteria,AAOB)是化能自养菌,由于其生理代谢的奇异性、细胞结构的特殊性以及对氮素循环的重要性,已成为环境工程、微生物以及海洋生物学等领域的研究热点.然而,AAOB未能实现纯培养的现状已成为AAOB代谢途径研究的巨大障碍.近年来兴起的宏基因组技术(Metagenomics)为AAOB代谢途径的研究提供了新手段.采用宏基因组技术,可直接研究微生物群体中某特定微生物基因组的结构与功能,摆脱了传统微生物学研究对纯培养的依赖,使未培养微生物的认识和开发成为可能.本文首先简述获取AAOB宏基因组信息的过程,然后通过比较由传统代谢研究方法和宏基因组技术获得的AAOB代谢途径的研究成果,论述基于宏基因组技术获得的对AAOB代谢的新理解,得出以下结果和结论:1)AAOB的碳素固定途径为乙酰辅酶A途径,碳素固定的还原力来自NADH或者QH2;2)AAOB氮素转化的重要中间产物是NO,而非NH2OH,并提出了以NO为核心的AAOB代谢的改进模型;3)AAOB的ATP合成途径为氧化磷酸化,推测的电子传递途径为N2H4—QH2—细胞色素bc1复合体;细胞色素bc1复合体再将电子用于NO2-还原和N2H4合成.AAOB的宏基因组技术使AAOB代谢途径的研究更具方向性.随着分子生物学理论和技术的不断发展,宏基因组学的升级技术(如宏转录组学、宏蛋白质组学)将为AAOB代谢途径的研究提供新的方法与平台.图3表1参51     

南雄优质适产烤烟因土施肥技术研究

采用田间试验方法,探明南雄县烟区三种土壤类型氮、磷、钾养分的供应特性,烤烟在各生育期吸收养分的规律,不同土壤类型氮、磷、钾肥的不同配比对烤烟产量和质量的影响。根据三年试验结果进行统计,提出优质适产烤烟因土施肥技术方法,为南雄烤烟生产的发展提供了科学的施肥依据。     

鼠Tcp11l1基因的结构与表达分析

为了分析鼠Tcp11l1基因的结构和表达,从小鼠睾丸组织总cDNA中扩增鼠Tcp11l1基因的开读框架(Open reading frame,ORF),定向克隆到真核表达载体pEGFP-N1,构建融合表达质粒pTcp11l1-EGFP,转染HEK293细胞,荧光显微镜下观察Tcp11l1基因的亚细胞定位;设计跨小鼠Tcp11l1基因两个外显子的引物,RT-PCR分析此基因在小鼠各组织中的表达情况.并利用生物信息学的方法对鼠Tcp11l1基因的结构进行初步预测.转染pTcp11l1-EGFP后在胞质中能明显看到绿色荧光信号,而在胞核和胞膜中无绿色荧光信号,表明鼠Tcp11l1蛋白定位于细胞质;RT-PCR分析结果表明,鼠Tcp11l1基因在小鼠各组织中广泛表达.生物信息学结果表明,鼠Tcp11l1和鼠Tcp11的蛋白具有相同的TCP11结构域,不能确定是否存在跨膜序列.鼠Tcp11l1基因和鼠Tcp11基因具有相似的亚细胞定位和TCP11结构域,表明这两个基因可能具有相似的受体功能.但是与Tcp11特异表达于睾丸组织的延长精细胞和精子不同,Tcp11l1为广泛表达,说明Tcp11l1可能在多种组织细胞中发挥作用.     

环境微生物中芳环加氧酶的获取策略

芳环加氧酶可催化芳香环的羟基化反应,广泛应用于生物修复及化工合成行业.本文在综述芳环加氧酶的分类和应用的基础上,详细探讨基于纯培养技术与宏基因组技术的芳环加氧酶开发策略,并首次对这些策略进行比较和评估.利用传统的纯培养技术已经获得大量的芳环加氧酶,然而这些加氧酶种类单一,重复率高,且新颖性不足.新兴的宏基因组技术赋予了开发未培养微生物资源的新思路,但利用其获取芳环加氧酶的研究刚刚起步,存在着筛选效率低、异源表达困难等难题.本文从基因信息量、筛选通量与效率、时间与成本以及目的基因下游表达可行性等方面对这两种方法进行综合评价,并提出宏基因组技术筛选不同类型芳环加氧酶的适用方法.纯培养技术与宏基因组技术的有机结合,与蓬勃发展的各种组学技术一起,将有助于推动芳环加氧酶的理论研究及生物催化产业的快速发展.     

深海沉积物微生物宏基因组文库中IMP环水解酶的筛选及基因克隆

为了解嘌呤合成途径的关键酶——IMP环水解酶的多样性,从深海沉积物样品中提取总DNA构建Cosmid宏基因组文库,从中筛选出一个具有IMP环水解酶活性的克隆(CAIMP1).通过基因步移的方法从CAIMP1的约35 kb的插入片段中扩增出长度为1 599 bp的完整的IMP环水解酶基因,该基因包含MGS和AICARFT_IMPCHas两个保守结构域.活性检测结果表明CAIMP1克隆的发酵液上清具有较强的IMP环水解酶活性.该酶与数据库中收录的IMP环水解酶的氨基酸序列同源性较低,系统发育分析也表明该酶与其它参照序列亲缘关系较远,表明该序列可能为一个新的IMP环水解酶基因.     

西藏米拉山高寒草甸土壤微生物DNA提取及宏基因组Fosmid文库构建

青藏高原土壤中富含特殊的微生物资源,因大多数未能得到培养而无法利用.提取微生物总DNA及构建宏基因组文库的方法是研究未培养微生物的重要方法(免培养法).两藏米拉山高寒草甸土壤中腐植酸、有机质含量非常高,DNA提取非常困难,本研究表明卣接法提取该样品的DNA片段小,杂质含量高,不能满足构建大插入片段文库的要求;结合低速离心和Nycodenz密度梯度离心先分离出微生物细胞的间接法虽然DNA产率降低,但是纯度高,片段长,多样性丰富,更适合于构建大插入片段文库.在间接法提取高质量西藏高寒草甸土壤微生物DNA的基础上,成功构建了一个包含30 624个克隆、库容量超过1 Gb、稳定性好的宏基因组Fosmid文库,为从西藏高原土壤中挖掘和利用新的功能基因研究奠定了基础.图7表3参27     

海洋微生物来源的天然产物开发研究进展

综述了近年来海洋微生物来源天然产物研究的新进展,总结了该类天然产物的开发策略.通过对样品采用不同的预处理方式、不同的分离培养基以及新的培养方法,讨论如何获得海洋来源的特有微生物和增加海洋来源微生物类群的多样性.阐述了在海洋微生物天然产物的开发过程中,采用宏基因组技术及基因组测序等手段,来发现难培养或不可培养微生物中的天然产物以及处于"沉默"状态的天然产物.最后介绍了异源生物合成、组合生物合成以及核糖体工程等技术在海洋微生物天然产物开发和改造中的应用,并举例论述了海洋微生物天然产物的开发.     

基于序列筛选发掘宏基因组文库中的新颖卤化酶基因

绝大部分微生物的不可培养性使微生物的开发利用受到了限制,而宏基因组学策略为研究土壤中的不可培养微生物提供了途径.天然卤化物具有抗菌活性和抗肿瘤活性等生物活性,卤化酶在催化化合物的卤化过程中,对化合物活性产生重要影响,而以卤化酶基因为探针,可以发现与之偶联的天然生物合成基因簇,为卤化物的发现提供基础.利用卤化酶基因序列的保守区域设计简并引物,筛选土壤宏基因组文库获得卤化酶阳性克隆,并对获得的卤化酶基因间的进化及其与天然产物合成的关系进行分析.结果显示:通过同源序列筛选获得了65个卤化酶阳性克隆,序列同源分析表明约85%的阳性克隆中的卤化酶基因序列与已知卤化酶的相似性低,所获卤化酶基因具有较好的新颖性和多样性.而对所获克隆中生物合成相关基因的分析表明其中一个克隆中同时存在聚酮合酶基因与卤化酶基因,其可能与卤代I型聚酮合成相关.本研究基于序列筛选的方法,从宏基因组文库中发现了新的卤化酶基因和聚酮合酶基因,为进一步发现新颖的天然卤化物生物合成基因簇及天然卤化物奠定了基础.     

Overcoming the inadequacy of single-criterion approaches to Life Cycle Assessment

In this work, the authors intend to introduce to the scientific community a novel integrated approach to environmental impact assessment, appropriately named SUstainability Multicriteria Multiscale Assessment (SUMMA). Its main objective is to overcome the inherent shortcomings of all single-criterion approaches, which constitute the vast majority of the Life Cycle Assessments performed to date in the scientific literature, and which invariably lead to partial and often misleading results. Furthermore, SUMMA is structured so as to enable a consistent sensitivity analysis of all the calculated impact indicators, a procedure that is of crucial importance for assessing the variability and reliability of the results, but which is only rarely adopted. The suggested framework is applied to selected case studies (coal gasification, bauxite mining, aluminium production and fuel cell manufacture), the results of which are discussed.     

An input-output analysis of environmental preservation

A well-established criterion for determining the growth potential of an economy, the technology of which can be represented by a nonnegative indecomposable square matrix, is examined critically. The concept of growth upon which this criterion is established is shown to be ambiguous since the environmental repercussions of economic activity are completely ignored. Assuming that this economy possesses an antipollution technology which has hitherto been ignored, an alternative criterion to gauge growth potential in the presence of complete environmental preservation is proposed. The magnitude of the uniform rate of profit which must obtain when the economy ignores the environment if a policy of environmental preservation is to be deemed technically feasible is calculated. Finally, a number of factors which would facilitate the implementation of a policy of environmental preservation are discussed.     

茂名小良桉树人工林生态经济效益分析与评价

利用模糊数学的方法,通过对小良的实地调杳和专家走访,挑选了30个对小良桉林生态经济影响较大的因子.建立了评价因子指标体系和五个评价等级,采用二个层次的综合评价模型对小良桉林生态经济效益首次进行了定量的综合评价.评价结果是:现在小良桉林生态经济效益为一般.总体态势是:16%为好,26%为较好,31%为一般.23%为较差,4%为差.综合评价得分为79.85分,介于一般和良好之间,这说明在小良桉树人工林的发展过程中存在许多显性的、潜在的问题,如果还不注意合理利用林地资源,就会使森林资源变为不可更新资源,不可持续利用.因此,科学客观地分析与评价小良桉林产业生态经济效益,为区域可持续发展提供科学依据和发展良策,实现生态和经济的综合调控具有现实意义.     

机动车污染模拟和地理信息系统支持

机动车污染是城市大气污染物的重要来源，对街区机动车污染排放量的预测和排放模拟对了解城市街区环境污染状况有重要价值。文章中将ＣＡＲ模式的模拟计算和ＧＩＳ集成在一起，通过ＶｉｓｕａｌＢａｓｉｃ进行了一个样例开发，其中ＧＩＳ功能由ＭａｐＸ控件实现。     

有机固体废弃物在持续农业中的资源化利用

有机固体废弃物产生量巨大且成分复杂,其对环境的污染已越来越多地引起人们的关注.它含有大量营养成分和有机质,在农业生产上除可以满足作物生长对养分的需要外,其丰富的有机质含量还可以改善土壤的物理化学性状,培肥土壤,有利于农业的可持续发展.有机固体废弃物的农业资源化处理利用是最积极有效的措施之一.     

Radionuclide diffuse in the environment

The purpose of this work is to relate the results and the conclusions which arose from the operation of nuclear plants from many years and from the application various radioecological models which describe the diffusing and the burdening of ecological system with radioactive substances in relation to the general problem of the pollution of the environment with whatever type of radioactive filthiness.

It is believed that the findings resulting from such comparison will be very interesting and will contribute to the taking of the right measures to face the pollution of the environment. This study is very important for countries such as Greece which has no nuclear plants but has a rapidly developed industry. The research has been mainly concentrated to the industrial area of Thessaloniki (Sindos) which is considered as the main source of diffused filthiness and which in the last few years has caused serious problems of pollution to the wider area of Thessaloniki.     

The optimal tax for maximum economic yield: Fishery regulation under rational expectations

This paper examines the optimal tax to achieve maximum economic yield (MEY) exploitation in a rational expectations model of a competitive open-access fishery. To analyze the dynamic evolution of resource use a structural model which explains the relationship between the firm and the industry is presented. The unregulated equilibrium is contrasted with the potential MEY. Conditions under which the unregulated equilibrium will be MEY are explored. In addition, a tax is devised which will cause non-MEY competitive exploitation to become MEY when the tax is implemented.     

Synergism between sib-rearing and sex ratio in Hymenoptera

Summary In many bees and wasps, solitary females produce offspring without help from other females. The transition from lone mothers producing offspring to situations in which females often help to rear siblings is an important step in the origins of complex sociality and nonreproductive castes. Recent work on Hymenoptera has stressed the role of sex ratio variation in this transition; when a mother's brood is more female biased than average, older daughters are favored to help rear their younger siblings because they are more closely related to sisters than to their own offspring. Here the direction of causality is from biased sex ratios, which arise by some extrinsic mechanism, to the origins of sib-rearing (eusociality). We present a model in which there is a synergism between sib-rearing and female-biased sex ratios, which may either complement the sex ratio variation idea by increasing the rate at which helping spreads or be an alternative hypothesis about the origins of eusociality. The synergism in our model depends on three conditions. 1) Daughters that help cause more food to be provisioned per offspring, which in turn causes larger offspring. 2) Females gain more than males by being large, which favors mothers with helpers to produce a higher proportion of daughters. 3) A helper's inclusive fitness rises as her mother's brood becomes increasingly female biased because a female helper is more closely related to her sisters than to her brothers. A female helper may also be more closely related to her sisters than to her own offspring, but this particular sibling-offspring relatedness asymmetry is not required by the synergism model. These three conditions create a synergism which favors a rapid transition from solitary (subsocial) to eusocial. Demographic and ecological factors that facilitate the evolution of eusociality reduce the stringency of the relatedness asymmetry condition (3) required by our idea. The synergism model therefore complements factors other than relatedness that may have been important during the evolution of eusociality.     

Stabilization-based soil remediation should consider long-term challenges

Soil remediation is of increasing importance globally, especially in developing countries. Among available remediation options, stabilization, which aims to immobilize contaminants within soil, has considerable advantages, including that it is cost-effective, versatile, sustainable, rapid, and often results in less secondary pollution. However, there are emerging challenges regarding the long-term performance of the technology, which may be affected by a range of environmental factors. These challenges stem from a research gap regarding the development of accurate, quantitative laboratory simulations of long-term conditions, whereby laboratory accelerated aging methods could be normalized to real field conditions. Therefore, field trials coupled with long-term monitoring are critical to further verify conditions under which stabilization is effective. Sustainability is also an important factor affecting the long-term stability of site remediation. It is hence important to consider these challenges to develop an optimized application of stabilization technology in soil remediation.     

A model system of four species with a unique equilibrium point

A four component model of two competing organisms, each with a specific parasite, has been exercised so as to investigate its equilibria. In addition to the equilibrium to which the model is known to return after moderate disturbance, severe perturbation led to a new steady state which had the appearance of an equilibrium but which was not maintained in the long term. Some discussion of what constitutes equilibrium and of stability of equilibria is presented.