利用广谱性和特异性组合诱变技术选育辅酶Q10高产菌

为提高辅酶Q10产量，采用了对细胞进行全面的非特异性诱变和针对特定途径的特异性诱变相结合的育种策略，以放射型根瘤菌(Rhizobium radiobacter)WSH2601为出发菌株，选择UV射线和亚硝基胍作为诱变剂，在筛选获得放线菌素D抗性突变株的基础上，通过进一步的诱变处理，分别获得了放线菌素D和L-乙基硫氨酸双抗性突变株、放线菌素D和维生素K双抗性突变株，以及抗放线菌素D和X-gal利用能力提高的突变株．与出发菌株相比，突变株辅酶Q10的产量提高幅度达25％-37％，其中一株放线菌素D和L-乙基硫氨酸双抗性突变株WSH-E25，胞内辅酶Q10含量和辅酶Q10总产量分别达到2．86mg g DCW^-1和40．0mg L^-1，均比出发菌株提高了37％，且遗传稳定性良好，图6表2参8     

通过原生质体技术提高泰乐菌素产生菌的产量

从泰乐菌素产生菌弗氏链霉菌０２８－３菌株出发，通过ＮＴＧ多次诱变，获得一株泰乐菌素产量提高２倍以上的突变株Ｈ１８８．再以原生质体再生和原生质作诱变后再生的手段，经过连续两轮处理，获得两株突变株：Ａ１１７和Ｄ８５．在最佳发酵培养基条件下，它们的泰乐菌素产量比Ｈ１８８分别提高６．５倍和５．５倍以上．     

克拉维酸产生菌的诱变育种

以克拉维酸产生菌棒状链霉菌Streptomyces clavuligerus CCRC11518 Ⅲ50为出发菌株,比较各种物理和化学诱变剂处理对其克拉维酸生物合成的影响,确定亚硝基胍为棒状链霉菌诱变育种的诱变剂及其处理剂量为2 mgmL-1、40 min.经业硝基胍处理40 min(处理浓度为2 mg mL-1)后,采用新颖理性化筛选方法,通过筛选自身代谢产物抗性突变株、克拉维酸抗性突变株和链霉素抗性突变株,最终得到一株克拉维酸高产菌Ⅵ118,其克拉维酸产量较出发菌株提高了67.9%.该高产突变株在琼脂斜面培养基上连续传接10代,克拉维酸产量保持稳定.     

温度和溶菌酶敏感菌株的L-谷氨酸合成

用温度敏感型菌株发酵生产L-谷氨酸不存在生物素亚适量问题,因此该方法在国际上被广泛使用.通常采用对出发菌株进行传统诱变的方法获得温度敏感型菌株.以谷氨酸棒杆菌CICC 10226为出发菌株,先克隆其ItsA基因,然后通过基因敲除的方法构建了突变菌株Corynebacterium glutamicum WT ΔL,该菌株同时具有温度敏感性和溶菌酶敏感性.经透射式电子显微镜观察发现,于38℃培养的突变株细胞与在30℃培养的同一种细胞相比,细胞明显增大,而出发菌株无该现象.发酵试验表明,在生物素过量的情况下,在发酵进入细胞产酸期后通过将发酵温度从原来的30℃提高到38℃,温度敏感突变株的产酸量增加近5倍.如果在发酵培养基巾添加适量的琥珀酸和乙酸,该菌株与不添加的在30℃培养的对照相比,产酸量增加近6.5倍.对于野生型出发菌株而言,在生物素过量的情况下,无论是否采用变温发酵方法都几乎不产酸.说明温度敏感突变株即使在生物素过量的情况下也能通过变温发酵诱导其合成并分泌产生L-谷氨酸.图10表1参13     

基于连续发酵驯化的高耐盐性酿酒酵母的育种

耐盐酿酒酵母菌株的育种对降低燃料乙醇生产成本具有重要意义.以具有优秀乙醇发酵能力的工业酿酒母菌株KF-7为出发菌株,通过连续发酵、产孢子及孢子培育、交配等获取稳定耐盐菌株.在盐胁迫条件下利用连续乙醇发酵驯化获得了耐盐突变菌株KF-7(4).在此基础上,通过孢子分离、培养、评价和交配,获得两株耐盐二倍体菌株KF-7(4)-3与KF-7-D1.这3株耐盐菌株在50次转接过程中保持着稳定的耐盐性.并且在9%KCl浓度下,3株耐盐菌株的乙醇发酵能力显著优于出发菌株KF-7:在15%YPD培养基中,发酵36 h时的乙醇浓度比出发菌株KF-7提高了21%.有盐和无盐条件下发酵过程中胞内海藻糖含量分析表明,突变菌株KF-7(4)和菌株KF-7(4)-3即使在无盐条件下的海藻糖积累能力明显高于出发菌株KF-7.本研究获得的变异酿酒酵母菌株具有较高的耐盐性和稳定性,耐盐性与胞内海藻糖积累能力提高相关.因此,基于连续发酵的进化工程手段可以有效地用于培育具有某种稳定性状的酿酒酵母菌株.     

卷曲霉素产生菌的改良

以卷曲霉素（ＣＰＭ）产生菌卷曲链霉菌Ｓ．ｃａｐｒｅｏｌｕｓ４００６（效价为６６４０ｕ／ｍＬ）为出发菌,经ＵＶ诱变／五氯酚浓缩,获得一株效价为７９５０ｕ／ｍＬ的营养缺陷型菌株４０６－３５,再经ＥＭＳ诱变,筛选到一株抗５０００ｕ／ｍＬＣＰＭ、效价为８５１３ｕ／ｍＬ的突变株５－４１,经原生质体ＮＴＧ诱变处理,获得一株效价为９０５０ｕ／ｍＬ的高产菌Ｎ１３菌株Ｎ１３连续传代１０代,卷曲霉素效价保持稳定通过单因子试验和多因子正交试验,确定了菌株Ｎ１３的最佳发酵条件菌株Ｎ１３在最佳发酵条件下卷曲霉素效价为９４８３ｕ／ｍＬ,比出发菌Ｓ．ｃａｐｒｅｏｌｕｓ４００６在原有发酵条件的效价提高４２８％     

利用原生质体诱变技术筛选脱落酸高产菌株

以葡萄孢属TB-3菌株为出发菌株制备其原生质体．在纤维素酶浓度为20g／L,蜗牛酶浓度为3g／L的酶解系统中,25℃酶解3h,其原生质体制备数可达67×105mL-1,在KYM上的再生率为46．1％,在KPDA上的再生率为33．6％,经过3轮原生质体紫外线诱变后回复再生,及对大量再生突变株进行发酵筛选,获得高产稳定株TB－3H8,其发酵液中ABA的质量浓度可达1．4g／L     

抗真菌多肽捷安肽素发酵条件的研究

对筛选出的一株芽孢杆菌ZK发酵产物抗真菌多肽捷安肽素的发酵培养基组分(碳源、氮源及无机盐)和工艺条件(发酵温度、起始pH,摇床转速,装液量)进行了摸索,通过单因素实验和正交优化实验,确定了ZK菌株发酵培养基最佳组成:生物氮素3.5％、葡萄糖3％、酵母膏0.08％、MgSO4·7H2O 0.5％、KH2PO4·3H2O 0.1％;最适温度为32℃;最适初始pH为8.0.在250 mL三角瓶中装50 mL培养基,于150 r min-1的旋转摇床上32℃振荡培养72 h,ZK菌株产捷安肽素的量达到最大.图8表5参14     

微波诱变选育耐酸高效厌氧产氢菌

为获得厌氧产氢菌的高效突变株,以产氢菌H-8为原始菌株进行微波诱变处理,对微波诱变参数进行了优化,考察了突变株的遗传稳定性、产氢特性及耐酸性.经过微波(低火)物理诱变得到5株高产氢突变株HW7、HW33、HW181、HW184和HW195,多次传代实验表明, HW195是稳定的高产突变株.突变株HW195具有较好的耐酸性,在pH值为2.8时仍能生长.通过间歇发酵实验,其最大产氢量和最大产氢速率分别达到2 460 mL/L培养基和340 mL L-1 h-1,比原始菌分别提高了50.7%和41.7%.图7表2参13     

化学诱变结合结构类似物法选育高苯丙氨酸解氨酶菌种

ＥＭＳ诱变结合结构类似物法提高含ＰＡＬ出发菌株红酵母Ｒｈｏｄｏｔｏｒｕｌａ ｓｐ．ＣＩＢＡＳＡ１４０１１００ 生物转化能力阳性率为３５．５ ％ ,部分突变株ＰＡＬ转化能力提高１ 倍以上,其中Ｅ１０５,Ｅ２４０ 具有快速转化能力,反应３ ｈＬＰｈｅ累积浓度达到１ ｇＬ左右．φ１０％ 甘油和３０ ｇＬＧｌｕ 对ＰＡＬ转化具有明显的稳定作用．用含φ１０％ 甘油和３０ ｇＬＧｌｕ 的反应液进行生物转化制备,Ｅ１０５ 和Ｅ２４０ 在２４ ｈ 期间ＬＰｈｅ 质量浓度大于２０ ｇＬ,转化率达８０ ％ ．     

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.     

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.     

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.     

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.     

Effects of Coffee Management on Deforestation Rates and Forest Integrity

Knowledge about how forest margins are utilized can be crucial for a general understanding of changes in forest cover, forest structure, and biodiversity across landscapes. We studied forest‐agriculture transitions in southwestern Ethiopia and hypothesized that the presence of coffee (Coffea arabica)decreases deforestation rates because of coffee's importance to local economies and its widespread occurrence in forests and forest margins. Using satellite images and elevation data, we compared changes in forest cover over 37 years (1973–2010) across elevations in 2 forest‐agriculture mosaic landscapes (1100 km² around Bonga and 3000 km² in Goma‐Gera). In the field in the Bonga area, we determined coffee cover and forest structure in 40 forest margins that differed in time since deforestation. Both the absolute and relative deforestation rates were lower at coffee‐growing elevations compared with at higher elevations (?10/20% vs. ?40/50% comparing relative rates at 1800 m asl and 2300–2500 m asl, respectively). Within the coffee‐growing elevation, the proportion of sites with high coffee cover (>20%) was significantly higher in stable margins (42% of sites that had been in the same location for the entire period) than in recently changed margins (0% of sites where expansion of annual crops had changed the margin). Disturbance level and forest structure did not differ between sites with 30% or 3% coffee. However, a growing body of literature on gradients of coffee management in Ethiopia reports coffee's negative effects on abundances of forest‐specialist species. Even if the presence of coffee slows down the conversion of forest to annual‐crop agriculture, there is a risk that an intensification of coffee management will still threaten forest biodiversity, including the genetic diversity of wild coffee. Conservation policy for Ethiopian forests thus needs to develop strategies that acknowledge that forests without coffee production may have higher deforestation risks than forests with coffee production and that forests with coffee production often have lower biodiversity value. Efectos de la Administración Cafetalera sobre las Tasas de Deforestación y la Integridad de los Bosques     

Globalization of the Cashmere Market and the Decline of Large Mammals in Central Asia

As drivers of terrestrial ecosystems, humans have replaced large carnivores in most areas, and human influence not only exerts striking ecological pressures on biodiversity at local scales but also has indirect effects in distant corners of the world. We suggest that the multibillion dollar cashmere industry creates economic motivations that link western fashion preferences for cashmere to land use in Central Asia. This penchant for stylish clothing, in turn, encourages herders to increase livestock production which affects persistence of over 6 endangered large mammals in these remote, arid ecosystems. We hypothesized that global trade in cashmere has strong negative effects on native large mammals of deserts and grassland where cashmere‐producing goats are raised. We used time series data, ecological snapshots of the biomass of native and domestic ungulates, and ecologically and behaviorally based fieldwork to test our hypothesis. In Mongolia increases in domestic goat production were associated with a 3‐fold increase in local profits for herders coexisting with endangered saiga (Saiga tatarica).That increasing domestic grazing pressure carries fitness consequences was inferred on the basis of an approximately 4‐fold difference in juvenile recruitment among blue sheep (Pseudois nayaur)in trans‐Himalayan India. Across 7 study areas in Mongolia, India, and China's Tibetan Plateau, native ungulate biomass is now <5% that of domestic species. Such trends suggest ecosystem degradation and decreased capacity for the persistence of native species, including at least 8 Asian endemic species: saiga, chiru (Pantholops hodgsoni), Bactrian camel (Camelus bactrianus), snow leopard(Panthera uncia), khulan(Equus hemionus), kiang (E. kiang), takhi (E. przewalski), and wild yak (Bos mutus). Our results suggest striking yet indirect and unintended actions that link trophic‐level effects to markets induced by the trade for cashmere. Globalización del Mercado de Cachemira y la Declinación de Mamíferos Mayores en Asia Central     

The lichen-rich coastal heath vegetation on the isle of Anholt, Denmark — conservation and management

The high conservational value of the lichen-rich vegetation and landscape of the marine foreland Ørkenen on the isle of Anholt is treated from a Danish as well as a European perspective. The sensitivity of the lichen-rich vegetation to physical disturbance is emphasized. The impact of invasive species such asPinus mugo as well as the effect of atmospheric deposition of nutrients on the heaths at Anholt is described. Considerations related to the development of a management plan for Ørkenen are presented.     

Behavior and ecology of Old World Luperini beetles of the genusAulacophora (Coleoptera: Chrysomelidae)

Summary The attraction of Old World leaf beetles in the genusAulacophora to kairomones and parakairomones which are effective lures for New WorldDiabrotica andAcalymma were investigated. Beetles captured on sticky traps baited with single and multicomponent lures were no different from the control traps for two species ofAulacophora. Yellow colored traps and squash blossoms are attractive toAulacophora beetles which detect sub-microgram quantities of cucurbitacins on silica gel. Leaf feeding behavior and flight activity data are correlated with varietal preference of threeAulacophora species. The common response byDiabrotica andAulacophora to cucurbitacins reinforces the two groups' coevolutionary association with the Cucurbitaceae. The apparent lack of a common response toCucurbita blossom volatiles suggests recent evolutionary pathways are substantially different for these two groups of beetles.     

Use of stochastic patch occupancy models in the California red-legged frog for Bayesian inference regarding past events and future persistence

Assessing causes of population decline is critically important to management of threatened species. Stochastic patch occupancy models (SPOMs) are popular tools for examining spatial and temporal dynamics of populations when presence–absence data in multiple habitat patches are available. We developed a Bayesian Markov chain method that extends existing SPOMs by focusing on past environmental changes that may have altered occupancy patterns prior to the beginning of data collection. Using occupancy data from 3 creeks, we applied the method to assess 2 hypothesized causes of population decline—in situ die-off and residual impact of past source population loss—in the California red-legged frog. Despite having no data for the 20–30 years between the hypothetical event leading to population decline and the first data collected, we were able to discriminate among hypotheses, finding evidence that in situ die-off increased in 2 of the creeks. Although the creeks had comparable numbers of occupied segments, owing to different extinction–colonization dynamics, our model predicted an 8-fold difference in persistence probabilities of their populations to 2030. Adding a source population led to a greater predicted persistence probability than did decreasing the in situ die-off, emphasizing that reversing the deleterious impacts of a disturbance may not be the most efficient management strategy. We expect our method will be useful for studying dynamics and evaluating management strategies of many species.