海洋微藻活体及乙醇固定状态下基因组DNA的微量提取方法

探讨了海洋微藻活体及固定状态下基因组DNA的提取方法,即作者改进的CTAB法.结果表明,CTAB改进法提取的基因组DNA蛋白质、酚、盐和小分子的污染较少,多糖成份也得到了有效去除.用5%乙醇固定样本提取DNA的效果最好,对原生动物抑制效果也较明显;10%以上的乙醇可杀死原生动物,但对DNA的降解率较大;30%以上的乙醇对细菌抑制较明显,但DNA都有较大程度的降解.本法提取的DNA可用于正常的酶切和PCR.用甲醛、甲醇/冰醋酸等其它固定液固定样品,DNA的得率低或者质量和纯度低.图7表1参27     

一种提取富含油脂植物组织总RNA的改良SDS酸酚法

采用常规的方法提取富含油脂的植物组织总RNA常难以取得满意的结果.对林莎两次裂解法进行了改良,即将SDS的浓度降低至1%,所用酸酚的pH值降低至4.0,去掉提取液Ⅱ处理步骤,获得的总RNA效果较好:得率高,RNA产量可达74.7 μg(100 mg)-1;没有DNA污染,A260 nm/A280 nm/2.026,A260 nm/A230 nm值为2.082,说明蛋白质及其它污染非常低,通过RT-PCR成功地扩增出了麻疯树curcin基因长为927 bp的特异条带.相比林莎的方法,改良SDS酸酚法具有经济、简便、适用性广的特点.图5表1参13     

从顽拗植物荔枝中提取基因组DNA技术的研究

针对荔枝体内含有大量影响ＤＮＡ提取质量的多酚等次生代谢物质的特点，在常规ＳＤＳ和ＣＴＡＢ提取方法的基础上做了技术改进，即在核裂解之间先破碎细胞，将存在于细胞质中次生物质除去后再裂解细胞核，同时加入活性炭以吸咐杂质反复清洗几次后加入核型解液释放基因组ＤＮＡ，纯化之后经外观和电泳检测，以及Ｄ２６０ｎｍ和Ｄ２８０ｎｍ比值的测定，限制性内切酶反应，ＰＣＲ扩增的结果表明，用改进方法提取的ＤＮＡ无论在纯度上还     

一种经济、简单的微生物基因组DNA的提取方法

获得一定浓度和纯度的DNA是进行分子生物学研究的基础。破解细胞壁与细胞膜是获得基因组DNA的前提，而蛋白质和核酸物质的分离是获得高质量DNA产物的关键。目前，主要采用的破壁方法有：冷冻研磨法、溶菌酶法、EDTA测等，这些方法一般采用复杂的裂解液体系，并借助蛋白酶K和RNA酶的帮助来获得高质量的抽提产物。由于细胞裂解体系不仅配制十分麻烦，而日部分药品有毒操作危险性大，此外部分药品及相关酶试剂价格昂贵。本文充分利用DNA在不同温度下自身可变性与复性的特性和在高盐与高温条件下蛋白质能够变性并沉淀。以无菌的SDS（c／c=20％）和NaCl（c/c=8％）的混合液作裂解体系，在沸水浴中破壁膜并使得部分的蛋白质变性和DNA变性并得到初步分离；随后在60℃和72℃水浴中使变性的DNA复性和重新凝聚，同时让RNA、蛋白质和细胞壁碎片等杂质降解或沉淀，从而获得高质量的DNA产物。     

湖流域土壤微生物基因组总DNA分离纯化及其质粒文库的初步构建

采用研磨／冻融和SDV蛋白酶K热处理以及CTAB处理等理化方法，直接从太湖流域土壤样品中抽提得到微生物总基因组DNA，所得粗DNA用透析袋法以及Nucleaotrap suspension DNA纯化试剂盒进行了纯化，纯化过的DNA能够满是常用限制性内切酶酶切、细菌16S rDNA通用引物和随机引物进行PCR扩增的要求，将所得纯品DNA经过EcoRI部分酶切之后与酶切并脱磷酸的pSK^ 空载体连接，再以大肠杆菌JM109为宿主细胞初步构建了土壤微生物基因组文库，图6参7     

土壤宏蛋白质组学蛋白质提取方法及其应用

土壤宏蛋白质组学在揭示土壤微生物功能、代谢与环境相互作用方面具有广阔的应用前景,但由于土壤样品的特殊性,土壤蛋白质提取步骤是限制土壤宏蛋白质组学大规模应用的瓶颈之一.本文从样品制备、提取方法、影响因素等方面综述了土壤蛋白提取方面的研究进展.一般来说,根据实验目的、蛋白种类及后续研究方法设计相应的分组成收集策略才能取得较好的提取效果.土壤总蛋白、胞内蛋白与胞外蛋白分别有不同的提取方法.总蛋白提取一般采用直接提取法;胞外蛋白不需要裂解;胞内蛋白提取方法有直接提取法和间接提取法等.裂解、浓缩、去除腐殖质的方法以及提取液、pH值的选择等也会影响提取效果.此外,简单介绍了土壤宏蛋白质组学的应用,并对今后的研究工作提出展望.表1参36     

质粒DNA小量提取法的改进

介绍了一种改进的小量提取质粒DNA的方法,该方法用NH4Ac和LiCl代替常规方法中苯酚和氯仿的抽提,有效去除了样品中RNA和蛋白质;用95%乙醇代替无水乙醇沉淀质粒DNA,降低了终产物中盐和多糖等杂质的含量,获得的质粒DNA质量好,产率高,能满足常规分子生物学实验的要求,如PCR、酶切、转化大肠杆菌以及植物遗传转化.图4表1参7     

真菌核酸的一种快速提取方法

在总结多种真菌DNA和RNA提取方法的基础上，发展了一种提取真菌核酸新方法，与其它方法相比，该方法具有快速、操作简便等特点，而且提取DNA和RNA步骤基本一致，用该方法提取的DNA完整、纯度高，可用于PR、限制性内切酶酶切和Southern杂交等分子生物学操作；提取RNA产量高，RNA无降解，纯度高，适用Northern和cDNA合成等分子生物学操作。     

土壤RNA提取方法研究进展

总RNA提取是进行实时荧光定量PCR、Northern杂交分析及cDNA文库建立等分子生物学实验研究的基础.提取出高质量高产量的土壤RNA已成为研究土壤微生物的关键步骤.综述了常用的几种土壤RNA提取方法,包括强变性剂法、Trizol法、CTAB法、SDS-Phenol法、酶解法以及试剂盒等方法.提取过程中化学试剂的选择及配比需依据不同的土壤类型,其中Trizol法在提取总RNA时应用广泛,再结合适当的物理破碎及生物学方法,可得到理想的RNA.试剂盒法提取土壤RNA方便快捷,纯度高但产量低,且对土壤具有选择性.最后提出了土壤RNA提取过程常见的一些问题和注意事项,为土壤RNA提取方法的选择提供依据.     

镉铜污染尾矿土中添加耐镉铜菌剂J5后微生物区系多样性的变化

采用酶解法和化学裂解法相结合的方法从安徽铜陵镉铜污染尾矿土中提取出土壤微生物基因组总DNA,并应用PCR-DGGE技术对比研究尾矿土中添加外源硫酸镉、硫酸铜和耐镉铜菌剂J5(Pseudomonassp.)后微生物区系多样性的变化.结果表明,尾矿土中微生物基因组总DNA分子量大小为21kb左右,未复垦的尾矿砂中微生物总DNA量较少,CdSO4处理后的尾矿土中微生物基因组总DNA量明显下降,而添加菌剂J5的尾矿土中微生物基因组总DNA量上升.DGGE电泳图谱进一步显示,尾矿土样中添加镉离子使图谱条带减少,分析表明尾矿土中的细菌种类数减少;而添加菌剂J5使图谱中条带明显增加,表明菌剂J5可提高尾矿土中土著微生物的活性和群落丰富度,可用于复合重金属污染尾矿区的生态恢复.     

Representation of the world's biophysical conditions by the global protected area network

Protected areas (PAs) are often implemented without consideration of already existing PAs, which is likely to cause an overrepresentation of certain biophysical conditions. We assessed the representativeness of the current PA network with regard to the world's biophysical conditions to highlight which conditions are underprotected and where these conditions are located. We overlaid terrestrial and marine PAs with information on biophysical conditions (e.g., temperature, precipitation, and elevation) and then quantified the percentage of area covered by the PA network. For 1 variable at a time in the terrestrial realm, high temperature, low precipitation, and medium and very high elevation were underrepresented. For the marine realm, low and medium sea surface temperature (SST), medium and high sea surface salinity (SSS), and the deep sea were underrepresented. Overall, protection was evenly distributed for elevation across the terrestrial realm and SST across the marine realm. For 2 variables at a time, cold and very dry terrestrial environments had mostly low protection, which was also the case for low SST and low and medium SSS across most depths for marine environments. Low protection occurred mostly in the Sahara and the Arabian Peninsula for the terrestrial realm and along the Tropic of Capricorn and toward the poles for the marine realm. Although biodiversity measures are of prime importance for the design of PA networks, highlighting biophysical gaps in current PAs adds a frequently overlooked perspective. These gaps may weaken the potential of PAs to conserve biodiversity. Thus, our results may provide useful insights for researchers, practitioners, and policy makers to establish a more comprehensive global PA network.     

Quantitative composition of the defensive secretion ofBledius species (Coleoptera: Staphylinidae: Oxytelinae) is adapted to naturally occurring predators

Summary The adaptation of defensive secretions to their target organisms was examined for the abdominal gland secretions ofBledius furcatus, B. spectabilis andB. arenarius. Therefore the target organisms of the secretion of theseBledius species (i.e. their predators) had to be identified. At the collection sites examined these were the earwigLabidura riparia, the antCataglyphis bicolor, the flyLispe candicans, different carabids of the generaPogonus, Dichirotrichus, Dyschirius, Bembidion andCalathus and the wading birdsHaematopus ostralegus andCalidris alba. The secretion of the abdominal glands contains the toxin ptoluquinone dissolved in either-dodecalactone and 1-undecene (B. furcatus andB. spectabilis) or in octanoic acid and octyloctanoate (B. arenarius). The ratio of these solvents is species-specific. Application experiments using some of the natural insect predators (L. riparia, C. bicolor, Pogonus, Di. gustavii, Dyschirius) revealed that these solvent ratios provided a more effective deterrent than other possible ratios. Thus by combining the solvents in certain ratios, the capability of cuticular penetration and therefore the effectiveness of the defensive secretions are adapted to their natural targets.     

Democracy versus development: New urban debates

The politics of development includes subtexts of choice, as underpinned by community consensus (or lack of it), against the larger backdrop of a liberal democracy, with its intricate power structures that influence the choice of concepts and often lead to complicity rather than consent. A dilemma exists between the understanding of field-level practitioners and that of academics, whose understandings of progress differ. Progress can (and often does) imply long-term change across generations whereas development is often more a matter of crisis management and delivering tangible results to end-users. This makes it a bone of contention across political systems, irrespective of local or global dynamics. Development can emerge as a tool to be wielded for power and further political mileage at the expense of progress. Progress cannot be studied as a counterpoint to development as the sense of history is often missing in development interventions. Development tends to be piecemeal as its very logic often stems from the rationale of power. This is not to suggest, however, that development is only about politics; it is definitely more than the politics of everyday life and involves all of civil society that requires its critical and immediate attention. In this sense, it has become a priority for politicians with an underlying agenda designed to out-manouevre dissent and all statements of protest.     

Achieving Open Access to Conservation Science

Conservation science is a crisis discipline in which the results of scientific enquiry must be made available quickly to those implementing management. We assessed the extent to which scientific research published since the year 2000 in 20 conservation science journals is publicly available. Of the 19,207 papers published, 1,667 (8.68%) are freely downloadable from an official repository. Moreover, only 938 papers (4.88%) meet the standard definition of open access in which material can be freely reused providing attribution to the authors is given. This compares poorly with a comparable set of 20 evolutionary biology journals, where 31.93% of papers are freely downloadable and 7.49% are open access. Seventeen of the 20 conservation journals offer an open access option, but fewer than 5% of the papers are available through open access. The cost of accessing the full body of conservation science runs into tens of thousands of dollars per year for institutional subscribers, and many conservation practitioners cannot access pay‐per‐view science through their workplace. However, important initiatives such as Research4Life are making science available to organizations in developing countries. We urge authors of conservation science to pay for open access on a per‐article basis or to choose publication in open access journals, taking care to ensure the license allows reuse for any purpose providing attribution is given. Currently, it would cost $51 million to make all conservation science published since 2000 freely available by paying the open access fees currently levied to authors. Publishers of conservation journals might consider more cost effective models for open access and conservation‐oriented organizations running journals could consider a broader range of options for open access to nonmembers such as sponsorship of open access via membership fees. Obtención de Acceso Abierto a la Ciencia de la Conservación     

A framework to measure the wildness of managed large vertebrate populations

As landscapes continue to fall under human influence through habitat loss and fragmentation, fencing is increasingly being used to mitigate anthropogenic threats and enhance the commercial value of wildlife. Subsequent intensification of management potentially erodes wildness by disembodying populations from landscape-level processes, thereby disconnecting species from natural selection. Tools are needed to measure the degree to which populations of large vertebrate species in formally protected and privately owned wildlife areas are self-sustaining and free to adapt. We devised a framework to measure such wildness based on 6 attributes relating to the evolutionary and ecological dynamics of vertebrates (space, disease and parasite resistance, exposure to predation, exposure to limitations and fluctuations of food and water supply, and reproduction). For each attribute, we set empirical, species-specific thresholds between 5 wildness states based on quantifiable management interventions. We analysed data from 205 private wildlife properties with management objectives spanning ecotourism to consumptive utilization to test the framework on 6 herbivore species representing a range of conservation statuses and commercial values. Wildness scores among species differed significantly, and the proportion of populations identified as wild ranged from 12% to 84%, which indicates the tool detected site-scale differences both among populations of different species and populations of the same species under different management regimes. By quantifying wildness, this framework provides practitioners with standardized measurement units that link biodiversity with the sustainable use of wildlife. Applications include informing species management plans at local scales; standardizing the inclusion of managed populations in red-list assessments; and providing a platform for certification and regulation of wildlife-based economies. Applying this framework may help embed wildness as a normative value in policy and mitigate the shifting baseline of what it means to truly conserve a species.     

Sexual Predators,Energy Development,and Conservation in Greater Yellowstone

Abstract: In the United States, as elsewhere, a growing debate pits national energy policy and homeland security against biological conservation. In rural communities the extraction of fossil fuels is often encouraged because of the employment opportunities it offers, although the concomitant itinerant workforce is often associated with increased wildlife poaching. We explored possible positive and negative factors associated with energy extraction in the Greater Yellowstone Ecosystem (GYE), an area known for its national parks, intact biological diversity, and some of the New World's longest terrestrial migrations. Specifically, we asked whether counties with different economies—recreation (ski), agrarian (ranching or farming), and energy extractive (petroleum)—differed in healthcare (gauged by the abundance of hospital beds) and in the frequency of sexual predators. The absolute and relative frequency of registered sex offenders grew approximately two to three times faster in areas reliant on energy extraction. Healthcare among counties did not differ. The strong conflation of community dishevel, as reflected by in‐migrant sexual predators, and ecological decay in Greater Yellowstone is consistent with patterns seen in similar systems from Ecuador to northern Canada, where social and environmental disarray exist around energy boomtowns. In our case, that groups (albeit with different aims) mobilized campaigns to help maintain the quality of rural livelihoods by protecting open space is a positive sign that conservation can matter, especially in the face of rampant and poorly executed energy extraction projects. Our findings further suggest that the public and industry need stronger regulatory action to instill greater vigilance when and where social factors and land conversion impact biological systems.     

Comparison of the insecticidal effects of water extracted and intact aromatic plants onAcanthoscelides obtectus,a bruchid beetle pest of kidney beans

Summary Acanthoscelides obtectus (Say) is one of the most damaging pests of kidney beans,Phaseolus vulgaris L. worldwide. However, aromatic plants from the families Lamiaceae, Lauraceae, Myrtaceae and Poaceae can protect kidney beans by direct or delayed insecticidal effect, through increased adult mortality and inhibition of beetle reproduction (both oviposition and adult emergence). The efficiency of hydrodistillated and intact plants fromThymus vulgaris andT. serpyllum, Mentha piperata, Rosmarinus officinalis, Satureia hortensis, Eucalyptus globulus, Laurus nobilis, Origanum vulgare, andCymbopogon nardus was compared. For both extracts,Origanum vulgare had the best effect. The insecticidal effect was induced by more than the essential oils because no significant difference was noticed between distilled and intact plants extract. Inhibition of reproduction was particularly important. These results suggest that lipidic as well as non lipidic, allelochemicals, such as phenolics, or non-protein amino-acids, or flavonoids may be involved in the toxicity of aromatic plants to this beetle.     

Rapid,recurring, structured survey versus bioblitz for generating biodiversity data and analysis with a multispecies abundance model

A bioblitz inexpensively and quickly generates biodiversity data, but bioblitzes are often conducted with haphazard, unreplicated sampling. Results tend to be taxonomically, geographically, or temporally biased, lack metadata, and consist of lists of observed taxa that do not enable further analyses or correction for imperfect detection. A rapid, recurring, structured survey (RRSS) uses a structured sampling design and temporal and spatial replication to survey randomly selected sites on a conservation property. We participated in a loosely structured bioblitz and a subsequent RRSS at Big Canoe Creek Nature Preserve in Springville (St. Clair County), Alabama (USA) to compare observed richness derived from the 2 survey approaches. The RRSS data structure enabled us to fit models that accounted for imperfect detection to estimate abundances, occupancy probabilities, and habitat associations. The loosely structured bioblitz data could not be used in such models. We present a new integrated multispecies abundance model that we applied to avian RRSS data. Our model extension enables estimation for the community, employs data augmentation to estimate the number of undetected species, and incorporates covariates. The RRSS generated a more comprehensive and less biased list of observed taxonomic richness than the loosely structured bioblitz (e.g., 73 vs. 45 bird species and 104 vs. 63 insect families from the RRSS vs. loosely structured bioblitz, respectively). Models fit to the RRSS data identified seasonal patterns in avian community composition and allowed for estimation of habitat–occupancy relationships for insect taxa. The RRSS protocol has potential for broad transferability as a standardized, quick, and inexpensive way to inventory biodiversity and estimate ecological parameters while providing an outreach opportunity.     

Compliance with and ecosystem function of biodiversity offsets in North American and European freshwaters

Land-use change via human development is a major driver of biodiversity loss. To reduce these impacts, billions of dollars are spent on biodiversity offsets. However, studies evaluating offset project effectiveness that examine components such as the overall compliance and function of projects remain rare. We reviewed 577 offsetting projects in freshwater ecosystems that included the metrics project size, type of aquatic system (e.g., wetland and creek), offsetting measure (e.g., enhancement, restoration, and creation), and an assessment of the projects’ compliance and functional success. Project information was obtained from scientific and government databases and gray literature. Despite considerable investment in offsetting projects, crucial problems persisted. Although compliance and function were related to each other, a high level of compliance did not guarantee a high degree of function. However, large projects relative to area had better function than small projects. Function improved when projects targeted productivity or specific ecosystem features and when multiple complementary management targets were in place. Restorative measures were more likely to achieve targets than creating entirely new ecosystems. Altogether the relationships we found highlight specific ecological processes that may help improve offsetting outcomes.     

Some polyphagous Homoptera gain protection from their natural enemies by feeding on the toxic plantsSpartium junceum andErythrina corallodendrum (Leguminosae)

Summary Interactions were studied among alkaloid-containing legumes (Erythrina corallodendrum andSpartium junceum) and non-toxic plants (Citrus sinensis, Cucurbita moschata andEuphorbia tirucalli), several polyphagous homopterans,Aphis craccivora (Aphididae),Icerya purchasi, I. aegyptiaca (Margarodidae),Lepidosaphes ulmi (Diaspididae) andPlanococcus citri (Pseudococcidae), and some major natural enemies of these homopterans. Significant reductions in survival due to negative effects of alkaloid containing as compared with non-alkaloidal plants were recorded for the predatorsRodolia cardinalis andChilocorus bipustulatus, but not forCryptolaemus montrouzieri (Coleoptera: Coccinellidae),Chrysoperla carnea (Neuroptera: Chrysopidae) andSympherobius sanctus (Neuroptera: Sympherobiidae). The development time of the larvae or pupae ofR. cardinalis, C. carnea andS. sanctus was longer on the toxic plants than on the non-toxic ones. The percentage of parasitism ofA. craccivora collected from the non-alkaloidal plantsVicia palaestina andMelilotus albus was much higher than that onS. junceum. The parasitoid complexes ofA. craccivora differed between both plant groups. The nutritive value of honeydew ofI. purchasi andA. craccivora, as expressed by the life span ofEncyrtus infelix (Hymenoptera: Encyrtidae) adults, was also investigated. Life spans were significantly longer when the wasps fed on honeydew produced on non-alkaloidal plants (C. sinensis andPittosporum tobira) than on alkaloid containing plants whenI. purchasi — but notA. craccivora — was the producer. It is suggested that the chemical defense ofE. corallodendrum andS. junceum is exploited by polyphagous phytophages to reduce predation. In nature, population growth and density of four of the investigated homopterans are conspicuously high when they developed on the alkaloid containing plant species, and very low on non-alkaloid plants. The efficiency of their natural enemies may be reduced by sequestration of alkaloids (or other toxic plant compounds) or their transfer into excreted honeydew. Therefore it is assumed that a generalist phytophagous homopteran may be protected from its natural enemies, although at different rates of efficiency, if it can safely sequester the host allelochemical when it develops on toxic species within its host range.