基于宏基因组学壳聚糖酶挖掘研究

壳聚糖酶(EC3.2.1.132)可有效地将壳聚糖降解为活性良好和应用前景广泛的低分子壳聚糖或壳寡糖,但目前存在专一性强的壳聚糖酶发酵水平低、种类单一、应用性能较差等问题.本研究从虾蟹堆积场淤泥采样,采用宏基因组文库方法,从其Fosmid文库中筛选出壳聚糖酶基因ChiE.测序表明,ChiE由1 362 bp组成,编码453个氨基酸,预测分子量(Mr)为42×103左右.将ChiE与pET-28a(+)载体连接,转化大肠杆菌Escherichia coli Rosetta-gami(DE3)构建了壳聚糖酶重组菌.采用离子交换柱(DEAE Sepharose FF)和凝胶色谱柱(SuperdexTM 75)纯化目的蛋白.酶学性质结果显示,该酶最适作用温度70℃,最适pH 6.0,Li+、Sr2+、K+等离子对其具有一定激活作用,Fe3+、Pb2+、Fe2+等离子则具有不同程度的抑制.在最适作用条件下,该酶比酶活为2 158 U/mg.本研究表明,重组菌E.coli Rosetta-gami(DE3)/ChiE具有培养简便、酶发酵产量大等优点,结果可为利用基因工程菌生产壳聚糖酶奠定基础.图5表2参17     

硫化叶菌病毒dUTPase基因的表达及酶学活性分析

对硫化叶菌病毒(Sulfolobus virus)STSV2脱氧尿苷焦磷酸酶(dUTPase)基因进行克隆表达,并分析其酶学特征.根据STSV2的dUTPase基因序列设计特异性引物,以病毒STSV2 DNA为模板进行PCR扩增,将产物克隆至原核表达载体pET32a(+)中,然后,将其转化至Escherichia coli BL21(DE3)进行dUTPase的IPTG诱导表达及纯化,通过SDS-PAGE检测其大小,并测定其酶学活性.结果表明,诱导表达的重组dUTPase蛋白分子量(Mr)约为38×103(含pET32a(+)自带的助溶标签),重组dUTPase在Mg2+存在的情况下能特异性催化dUTP生成dUMP和PPi,其反应最适温度为60℃,在pH值3-8之间有较高的活性,其在提高PCR扩增效率和特异性方面具有一定的作用.本研究通过克隆表达获得了硫化叶菌病毒(Sulfolobus virus)STSV2脱氧尿苷焦磷酸酶,其在基因扩增领域有一定的应用价值.图5表1参18     

蕹菜类囊体膜磷酸酯酶的分离纯化和部分性质

使用NaCl抽提、硫酸铵分步沉淀、离子交换和疏水柱层析等方法 ,从蕹菜叶绿体类囊体膜中分离纯化到一种蛋白磷酸酯酶 .SDS -PAGE检测表明 ,这种磷酸酯酶的分子量 (Mr)为 14 .9× 10 3 .该酶具有水解磷酸单酯类物质的活性 ,水解pNPP的Km值为 1.76× 10 -6mol/L ;体外测活时最适pH为 5 ,属于酸性磷酸酯酶 ;该酶耐热 ,在 5 0℃时活力达到最高 ,对NaCl不太敏感 ;但EDTA和NaF可以明显影响该酶的活性 .图 6表 3参 2 4     

耐辐射奇球菌RecR和SSB蛋白的克隆表达及抗紫外功能

RecR和SSB蛋白是耐辐射奇球菌(Deinococcus radiodurans)同源重组修复RecFOR途径的关键蛋白.以耐辐射奇球菌基因组为模板,PCR扩增recR和ssb基因片段,以质粒pET32a(+)为载体,构建重组质粒pET32a(+)-recR和pET32a(+)-ssb,并转化到Escherichia coli BL21(DE3)中,IPTG诱导蛋白表达,表达产物采用SDS-PAGE鉴定,同时,应用平板菌落计数法检测紫外辐照前后各组细菌生存率变化,研究RecR和SSB蛋白在E.coli BL21(DE3)抗紫外线辐射中的作用.结果表明,RecR和SSB能够克隆至pET32a(+)载体并在E.coli BL21(DE3)中诱导表达.紫外辐照生存率实验结果提示,RecR和SSB蛋白能增强E.coli BL21(DE3)对紫外线辐射的抵抗能力.RecR和SSB的成功表达及紫外抗性特点将为耐辐射奇球菌超强耐辐射机制的研究提供实验基础.     

乙酰辅酶A合成酶的酶学性质及其在甲羟戊酸合成中的应用

乙酰辅酶A合成酶(ACS)在微生物体内可直接将乙酸转化为乙酰辅酶A,乙酰辅酶A再进入微生物的碳代谢循环过程.为提高乙酸的利用效率,筛选催化活性较高的乙酰辅酶A合成酶,从大肠杆菌(Escherichia coli K-12)和巴氏醋酸杆菌(Acetobacter pasteurianus ATCC 33445)中扩增得到3个不同的乙酰辅酶A合成酶基因,将这3个基因分别连接到p ET-28a(+)载体上,Western Blot分析表明,3个基因均在E.coli BL21(DE3)中成功表达.对3个不同来源的乙酰辅酶A合成酶的酶活性进行比较,发现来自于巴氏醋酸杆菌的乙酰辅酶A合成酶(ACS2)具有较高的催化活力;其最适温度37℃,最适pH在7-8之间;在最适反应条件下,K_m为1.16 mmol/L,最大反应速度(V_(max))为20.45 mmol L~(-1) min~(-1).将ACS2应用到以乙酸为底物生物合成甲羟戊酸过程中,当过表达ACS2时,单批补料发酵条件下,甲羟戊酸产量达到6.59g/L.本研究筛选到1种催化活性较高的乙酰辅酶A合成酶,显著提高了甲羟戊酸的生物合成,可为乙酰辅酶A合成酶的进一步研究和应用提供理论支持.(图8表1参18)     

朱砂叶螨乙酰胆碱酯酶的原核表达及其活性分析

乙酰胆碱酯酶(ACh E)在神经信号传导过程中起重要作用,是氨基甲酸酯类和有机磷类农药的主要作用靶标.对朱砂叶螨ACh E进行体外表达和纯化,并分析其活性.将朱砂叶螨ace基因序列插入到原核高效表达载体p ET-30a中,构建表达载体p ET-30a/ace,转入表达菌株Escherichia coli BL21(DE3)中,IPTG诱导高效表达;镍柱纯化目的蛋白,经SDS-PAGE和Western Blot鉴定目的蛋白;以纯化的螨ACh E为抗原制备抗螨ACh E特异性抗体;改良的Ellman法测定螨ACh E活性.结果获得了相对分子质量(Mr)约为68×103的较高纯度的螨ACh E蛋白,检测大部分为可溶性表达,酶活检测具有乙酰胆碱酯酶活性,并制备了107效价的抗螨ACh E特异性抗体.采用重组ACh E(IC50=5.4 mmol/L)检测毒扁豆碱抑制活性比螨粗酶液(IC50=58.7 mmol/L)灵敏度提高11倍.本研究构建了螨ACh E体外高效表达载体并获得了具有较好活性的重组螨ACh E,可为抗ACh E杀螨剂的体外高通量筛选和以ACh E为靶标的农药残留检测奠定基础.     

维生素C二步发酵中L-山梨糖脱氢酶的性质及作用

从氧化葡萄糖酸杆菌细胞质中分离纯化出L 山梨糖脱氢酶 (SDH) ,其分子量为 46× 10 3 ,表观分子量为 190× 10 3 ;在测试范围内 ,最适pH是 7.4,温度为 5 5℃ ,最稳定的 pH是 7.0 ,温度为 30℃以下 ;FeCl3 促进酶活 ,CoCl2 抑制酶活 .该酶活力与发酵产物 2 酮基 L 古龙酸的合成呈正相关 ;伴生菌促进产酸菌生长和代谢 ,并使该酶比活力增加 ,从而提高发酵系统中该酶的总活力 .图 11表 5参 9     

地衣芽孢杆菌生麦芽糖α-淀粉酶的基因克隆与鉴定

以地衣芽孢杆菌ATCC14580基因组DNA为模板,PCR扩增出1.7 kb大小的麦芽糖淀粉酶基因amyM,克隆入表达载体pET-28a(+),转化Escherichia.coli BL21(DE3),经IPTG诱导,测定麦芽糖淀粉酶酶活性.结果表明,麦芽糖淀粉酶基因amyM获得了活性表达,酶活力为3.797 U/mL,SDS-1PAGE电泳结果显示出相对分子质量约为67×103的特异性蛋白质条带.酶学性质分析表明,重组麦芽糖淀粉酶的最适反应温度为45℃,最适反应pH为6.5,在45℃以下的中低温环境保持稳定,且能在比较宽的pH范围内(pH 4.5～8.5)稳定.图3表1参16     

邻苯二酚2,3-双加氧酶基因克隆、定位和高效表达

采用特异性引物，以菲、芘降解菌株ZL5的代谢性质粒为模板，扩增出邻苯二酚2，3—双加氧酶(C23O)基因，将该基因和表达载体pET—30a( )连接，转化E.coli JM109(DE3)，获得了高效表达的转化子，SDS—PAGE结果表明，转化子的C23O蛋白不仅在细胞内存在，而且能被分泌到胞外，薄层扫描显示，转化子细胞内和细胞外表达蛋白总量占细胞总蛋白的42％，酶活分析表明，分布在转化子细胞内、外的表达蛋白都具有较高的C23O比活力，Southern杂交将菌株ZL5的C23O基因定位在内生质粒的不同酶切片段上。图5表1参12。     

烯酮还原酶基因的克隆与优化表达

烯酮/酯还原酶可以专一性地还原烯酮/酯中的碳碳双键,并引入两个手性中心,是手性化合物生物催化合成的重要酶类之一;在使用烯酮/酯还原酶进行全细胞手性分子生物催化合成中,其他还原酶的存在常导致副反应的产生.为研究烯酮/酯还原酶的理化性质、底物的专一性、产物的手性特点等,需要经过纯化的烯酮/酯还原酶.为此,根据烯酮还原酶(Enoate reductase,ERs,EC 1.3.1.31)的基因序列设计引物,以丙酮丁醇梭菌(Clostridium acetobutylicum)ATCC824基因组DNA为模板,通过PCR扩增技术得到了目标酶的基因,目的片段全长为1 995 bp,共编码664个氨基酸,相对分子质量为73×103.将烯酮还原酶基因连接到pET-32a(+)上,构建表达质粒pET-32a(+)-ERs,并成功在Escherichia coli Rosetta(DE3)pLysS中表达.在摇床上优化的表达条件为:诱导温度为18℃,诱导起始时菌体D600 nm为0.6～0.8,转速为100 r/min,诱导剂IPTG浓度为0.1 mmol/L,诱导培养时间为12 h,表达的烯酮还原酶大部分以可溶性形式存在于菌体内.     

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.     

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.     

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     

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.