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     

重组人白血病抑制因子的表达、包涵体复性、纯化及其活性

白血病抑制因子(LIF)是一种在生物体内具有独特活性和重要调节作用因而广泛应用的多功能细胞因子.利用基因工程技术表达重组人白血病抑制因子(Recombinant human leukemia inhibitory factor,rhLIF)并进行包涵体的复性及纯化,将rhLIF基因克隆到原核表达载体pET32a中,成功构建重组hLIF基因工程菌,并研究该菌株发酵条件和纯化工艺.先将种子液进行发酵培养,诱导表达,收集诱导后的细胞;对细胞破碎,收集包涵体沉淀,洗涤包涵体;用6 mol/L盐酸胍变性重溶包涵体,镍离子亲和层析柱上直接复性及纯化,并用阴离子交换进一步纯化,复性率为75%,纯度达97%.结果显示基因序列与理论序列一致,SDS-PAGE分析结果表明其表达的外源蛋白质分子量与预期的目的蛋白质相对分子量大小相一致,均为35×10~3;Western blotting、MTT活性检测结果表明,此重组hLIF融合蛋白具有良好的抗原性和免疫原性.本研究成功建立了rhLIF原核表达、复性和纯化体系,可为进一步研究LIF生物学活性及产品开发提供试验基础.     

鹤望兰基因组DNA的提取方法

由于鹤望兰组织内含有大量多糖类及多酚类物质，严重干预DNA的抽提，利用常规的SDS法，CTAB法和高盐低pH值法都难以抽提出高质量的鹤望兰基因组DNA，本文报道了在进行若干预处理之后，再用常规的SDS，CTAB，SDS裂解的高盐低pH值法和CTAB裂解的高盐低pH值法提取基因组DNA的改进方法，根据外观，琼脂糖凝胶电泳检测，D260nm/D280nm比值的测定。限制性酶切反应，PCR扩增的结果表明，用改进方法提取的基因组DNA无论在纯度上还是在完整性上都比常规方法要好，其中改进SDS裂解的高盐低pH值法是提取鹤望兰基因组DNAS的最好方法。     

濒危植物厚朴SSR引物筛选及反应体系优化

探索适宜厚朴(Magnolia officinalis)简单重复序列(simple sequence repeat,SSR)-聚合酶链式反应(polymerase chain reaction,PCR)的最佳条件.在SSR引物筛选基础上,利用正交试验设计对影响PCR反应的5个因素(模板DNA、引物、Mg2+、dNTPs和Taq酶催化活性浓度)进行4个水平的优化,并通过温度梯度试验优化引物退火温度.在厚朴及其近缘物种的70对引物中,筛选出13对扩增条带清晰、多态性丰富的SSR引物.PCR各因素在不同水平下对反应体系的影响由大到小依次为引物浓度、Taq酶催化活性浓度、Mg2+浓度、dNTPs浓度和模板DNA浓度.PCR最佳优化体系:25 μL体系中,模板DNA质量浓度为2 ng·μL-1,引物浓度为0.6μmol·L-1,Mg2+浓度为2.0 mmol·L-1,dNTPs浓度为0.5 mmol·L-1,Taq酶催化活性浓度为0.03 U·μL-1(1U=16.67 nkat).最佳扩增程序:94℃预变性4 min,94℃变性30 s,48.0～59.0℃(退火温度随引物不同而定)退火30 s,72℃延伸1 min,35个循环后72℃延伸10 min.上述结果可为利用SSR-PCR技术研究濒危厚朴群体遗传学和分子生态学提供技术参数.     

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。     

从麻疯树胚乳中提取总RNA的快速方法

以麻疯树胚乳为材料,建立了一种适合于提取富含油脂、蛋白及多酚、多糖等次生物质的植物总RNA的方法.其中,提取液Ⅰ在1.5%SDS和400 mmol/L NaCl盐溶液条件下能快速有效破壁,酸酚/氯仿促进了细胞裂解以及油脂、蛋白的分离.而基于异硫氧酸胍法的提取液Ⅱ进一步强烈抑制Rnase的活性,确保RNA不被降解,并以5 mol/L高浓度NaCl溶液除去植物组织提取液中的多糖、多酚等次生物质,得到质量较好的总RNA样品.     

城市污水厂（A／O工艺）微生物群落结构及其动态变化

为了研究城市污水厂(A/O工艺)活性污泥中微生物群落结构及其动态变化,分别在运行不同时期从曝气池中提取活性污泥,通过细胞裂解直接提取活性污泥中的细菌基因组DNA,以细菌16S rDNA通用引物F357/R518,对活性污泥中提取的细菌基因组进行扩增,长约230 bp的扩增产物经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的DNA特征指纹图谱.结果显示,城市污水厂(A/O工艺)活性污泥中的微生物群落非常丰富,在不同时期存在一些各自的特有种属和共有种属,细菌群落结构的演替与系统负荷存在一定的相关性.整体来说,微生物群落演替不明显,微生物群落相似性较高,群落结构较为稳定.     

铈对马尾松离体胚DNA、RNA及蛋白质合成的影响

用含30mgL^-1CeCl3的培养瘁培养马尾松离体胚，采用^3H-T、^3H-U和^3H-Leu标记技术测定：结果表明，CeCl3明显提高马尾松离体胚在培养过程中对DNA、RNA和蛋白质前体的吸收，以及合成这些生物大分子的能力。CeCl3处理在15h开始迅速吸收DNA前体和合成DNA，比CK提前5h。CeCl3处理在10h开始迅速吸收RNA前体和合成RNA，比CK提前5h，CeCl3处理5h,蛋白质合成前体的吸收量和蛋白质合成量明显高于CK。图6参19     

RAPD扩增中商品酶体系对扩增产物的影响

随机选用了10条RAPD引物，采用3种商品TapDNA聚合酶体系，以5个不同物种的动植物总DNA为模板，进行RAPD扩增，实验发现，在对同一模板、采用同一引物进行的RAPD扩增中，仅仅因为使用了不同的商品TaqDNA聚合酶，获得的扩增产物差别极大；不同商品来源的PCR反应缓冲体系，对扩增产物也有一定影响，但相对较小，这说明，不同的商品酶体系对RAPD扩增产物影响很大，因此，影响RAPD扩增产物稳定性的一个关键因素是商品TaqDNA聚合酶本身；研究中前后一致地使用同一商品TaqDNA聚合酶体系，是成功进行RAPD分析的基础。     

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     

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     

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.     

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.     

Sequestration of cucurbitacin analogs by New and Old World chrysomelid leaf beetles in the tribe Luperini

Summary Two South American polyphagous leaf beetles,Diabrotica speciosa andCerotoma arcuata, selectively accumulated the bitter tasting compound 23,24-dihydrocucurbitacin D in their body after ingesting root tissues of cucurbit plants. Similarly, three Asian Cucurbitaceae-feeding specialists in the genusAulacophora were found to sequester the same compound. Cucurbitacin analogs were shown to deter feeding by a bird predator, indicating an allomonal role for these compounds in cucurbitacin-associated chrysomelid leaf beetles both of New and Old Worlds. The strong affinity to cucurbitacins, selective sequestration of the analogs and consequent protection from predators suggested an ecological adaptation mechanism developed in common among these two geographically isolated subtribes in the Luperini.     

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.     

Effects of Controlled Fire and Livestock Grazing on Bird Communities in East African Savannas

Abstract: In East Africa fire and grazing by wild and domestic ungulates maintain savannas, and pastoralists historically set fires and herded livestock through the use of temporary corrals called bomas. In recent decades traditional pastoral practices have declined, and this may be affecting biodiversity. We investigated the effects of prescribed fires and bomas on savanna bird communities in East Africa during the first and second dry seasons of the year (respectively before and after the rains that mark the onset of breeding for most birds). We compared abundance, richness, and community composition on 9‐ha burned plots, recently abandoned bomas, and control plots in the undisturbed matrix habitat over a 3‐year period. Generally, recently burned areas and abandoned bomas attracted greater densities of birds and had different community assemblages than the surrounding matrix. The effects of disturbances were influenced by interactions between primary productivity, represented by the normalized difference vegetation index, and time. Bird densities were highest and a greater proportion of species was observed on burned plots in the months following the fires. Drought conditions equalized bird densities across treatments within 1 year, and individuals from a greater proportion of species were more commonly observed on abandoned bomas. Yearly fluctuations in abundance were less pronounced on bomas than on burns, which indicate that although fire may benefit birds in the short term, bomas may have a more‐lasting positive effect and provide resources during droughts. Several Palearctic migrants were attracted to burned plots regardless of rainfall, which indicates continued fire suppression may threaten their already‐declining populations. Most notably, the paucity of birds observed on the controls suggests that the current structure of the matrix developed as a result of fire suppression. Traditional pastoralism appears critical to the maintenance of avian diversity in these savannas.     

A palaeo-hydrogeological model for arsenic contamination in southern and south-east Asia

An argument is presented in which areas of natural arsenic contamination of modern groundwaters throughout Asia have a common origin. Arsenic originally accumulated in oceanic ferro-manganoan sediments of the eastern Palaeo-Tethys. This was further concentrated through oceanic crustal extinction in what later became the south-east Chinese accreted mineralised terrain. Proto-Himalayan uplift of this area created the palaeo-drainage systems of the Ganges – Brahmaputra, Irrawaddy, Mekong, and Red Rivers, with consequent headwater erosion of arsenic-rich sediments. Their downstream deposition as immature and easily redistributed Neogene sandstones, silts, and iron-rich clays has created secondary and tertiary reservoirs of adsorbed and authigenic arsenic, from which the current arsenic-rich groundwaters have evolved. Considering river basins within the above palaeo-hydrogeological framework provides a basis for assessing the risk of arsenic in groundwater basins of south and south-eastern Asia.     

Threats to intact tropical peatlands and opportunities for their conservation

Large, intact areas of tropical peatland are highly threatened at a global scale by the expansion of commercial agriculture and other forms of economic development. Conserving peatlands on a landscape scale, with their hydrology intact, is of international conservation importance to preserve their distinctive biodiversity and ecosystem services and maintain their resilience to future environmental change. We explored threats to and opportunities for conserving remaining intact tropical peatlands; thus, we excluded peatlands of Indonesia and Malaysia, where extensive deforestation, drainage, and conversion to plantations means conservation in this region can protect only small fragments of the original ecosystem. We focused on a case study, the Pastaza‐Marañón Foreland Basin (PMFB) in Peru, which is among the largest known intact tropical peatland landscapes in the world and is representative of peatland vulnerability. Maintenance of the hydrological conditions critical for carbon storage and ecosystem function of peatlands is, in the PMFB, primarily threatened by expansion of commercial agriculture linked to new transport infrastructure that is facilitating access to remote areas. There remain opportunities in the PMFB and elsewhere to develop alternative, more sustainable land‐use practices. Although some of the peatlands in the PMFB fall within existing legally protected areas, this protection does not include the most carbon‐dense (domed pole forest) areas. New carbon‐based conservation instruments (e.g., REDD+, Green Climate Fund), developing markets for sustainable peatland products, transferring land title to local communities, and expanding protected areas offer pathways to increased protection for intact tropical peatlands in Amazonia and elsewhere, such as those in New Guinea and Central Africa which remain, for the moment, broadly beyond the frontier of commercial development.     

What Every Conservation Biologist Should Know about Economic Theory

Abstract: The last century has seen the ascendance of a core economic model, which we will refer to as Walrasian economics. This model is driven by the psychological assumptions that humans act only in a self‐referential and narrowly rational way and that production can be described as a self‐contained circular flow between firms and households. These assumptions have critical implications for the way economics is used to inform conservation biology. Yet the Walrasian model is inconsistent with a large body of empirical evidence about actual human behavior, and it violates a number of basic physical laws. Research in behavioral science and neuroscience shows that humans are uniquely social animals and not self‐centered rational economic beings. Economic production is subject to physical laws including the laws of thermodynamics and mass balance. In addition, some contemporary economic theory, spurred by exciting new research in human behavior and a wealth of data about the negative global impact of the human economy on natural systems, is moving toward a world view that places consumption and production squarely in its behavioral and biophysical context. We argue that abandoning the straightjacket of the Walrasian core is essential to further progress in understanding the complex, coupled interactions between the human economy and the natural world. We call for a new framework for economic theory and policy that is consistent with observed human behavior, recognizes the complex and frequently irreversible interaction between human and natural systems, and directly confronts the cumulative negative effects of the human economy on the Earth's life support systems. Biophysical economics and ecological economics are two emerging economic frameworks in this movement.