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1.
来自嗜热脂肪土芽孢杆菌的木聚糖内切酶XT6在工业上有着重要的应用,已经成功应用于工业规模的生产试验.本文作者在合成XT6基因全序列的同时对其密码子进行了优化,且构建重组质粒在大肠杆菌中高表达.通过优化表达条件,功能正常的XT6基因在大肠杆菌中成功过量表达,蛋白表达量占细胞中总蛋白的65%.重组表达的木聚糖内切酶XT6特性和天然酶相似,以桦木木聚糖为底物测定细胞提取物中木聚糖酶活性,最大活性高达3 030 U/mL.本文首次报道了来自嗜热脂肪土芽孢杆菌中木聚糖酶基因全序列的合成和在大肠杆菌中成功过量表达.图6表1参19  相似文献   

2.
用PCR方法从地衣芽孢杆菌 2 70 9和 6 816中扩增了碱性蛋白酶基因 (apr1和apr2 ) ,扩增的DNA片段插入到大肠杆菌载体 pET -2 8a中 ,构建成重组分泌型表达载体pAPR1、pAPR2 .pAPR1、pAPR2中碱性蛋白酶基因在大肠杆菌宿主JM 10 9(DE3)中得到表达 .SDS -PAGE分析显示融合表达产物的分子量均为 30 .5× 10 3 ,同核酸序列测定所推导的值相符 .表达产物分别占细胞总蛋白的 8.0 %和 7.5 % .2 70 9重组菌所得酶活为 12 10u/mL ,6 816重组菌所得酶活为 1175u/mL .研究发现 ,重组的碱性蛋白酶在进入大肠杆菌周质空间时可能存在前肽自动脱落的现象 .同时 ,对地衣芽孢杆菌 2 70 9碱性蛋白酶基因序列进行了测定和比较分析 ,发现与地衣芽孢杆菌 6 816碱性蛋白酶基因的同源性为 98% .图 5参 11  相似文献   

3.
构建了高效表达耐高温α-淀粉酶的枯草芽孢杆菌表达系统,并研究了该表达系统的主要特点.通过PCR扩增由地衣芽孢杆菌的基因组DNA分离高温淀粉酶基因后,将其克隆到质粒pSG703中.然后用pSG703质粒转化含温和性噬菌体φ105MU331的枯草芽孢杆菌,通过同源重组使高温淀粉酶基因插入到溶源性枯草芽孢杆菌的染色体上,并处于φ105MU331的强启动子下游.由于高温淀粉酶基因处于噬菌体的强启动子下游,在热诱导后可以实现高温淀粉酶的高效表达,诱导后8h内分泌到培养液中的高温淀粉酶活性可以达到9.58×103u/mL.本文构建的重组高温淀粉酶表达系统具有稳定、高效和杂蛋白分泌少的特点.图5参16  相似文献   

4.
专性自养极端嗜酸性氧化硫硫杆菌缺乏EMP、ED途径以及三羧酸循环的关键酶 (如磷酸果糖激酶等 ) ,不能氧化有机物获得能量 .本文利用PCR技术扩增E .coliK 12磷酸果糖激酶 1基因 (pfkA) ,将该基因与广泛寄主的IncQ质粒pJRD2 15连接构建重组质粒pSDK 1,该质粒可与pfk基因缺陷株E .coliDF10 10互补 .通过接合转移的方式将其导入氧化硫硫杆菌Tt 7中并得到表达 .pSDK 1在宿主Tt 7中有较好的稳定性 ,在无选择压力条件下连续传 5 0代仍可保留 75 % .酶活性测定表明 ,pSDK 1的pfkA基因在缺陷株E .coliDF10 10中表达水平〔(96 .6± 0 .6 6 )U/g蛋白〕略高于原始菌株E .coliK 12〔(85 .9± 1.12 )U/g蛋白〕 ;而在氧化硫硫杆菌中则以较低水平进行表达〔(12 .4± 0 .73)U/g蛋白〕 ,并且其表达水平不受培养基中葡萄糖的影响 .实验表明 ,在无机盐培养基中加入葡萄糖时 ,含质粒pSDK 1的氧化硫硫杆菌Tt 7可利用培养基中的葡萄糖而加快生长 ,而对照菌株的生长则未受明显影响 ;但是重组菌利用葡萄糖的速度较缓慢 .图 5表 2参 16  相似文献   

5.
与其它微生物相比,用大肠杆菌进行发酵生产乳酸有许多优势,但大肠杆菌没有L乳酸脱氢酶基因,不能利用糖发酵生产L乳酸.本文采用PCR技术,以乳酸片球菌(Pediococcusacidilactici)基因组DNA为模板,克隆得到L乳酸脱氢酶(Llactatedehydrogenase)基因,将该基因的ORF连接到表达质粒pET22b( ),获得重组质粒pETldhL.将pETldhL质粒转化大肠杆菌BL21株,实现了ldhL基因的表达;对含有ldhL基因的重组菌株进行表达研究,在30℃以IPTG诱导8h后,SDSPAGE分析可见明显特异性条带,酶活力达到6.49u/mL,是野生菌酶活力(2.82u/mL)的2.3倍.对重组表达的L乳酸脱氢酶生物学特性研究显示,它们的最适反应温度为27~30℃,最适反应pH为5.0~5.3.图5参15  相似文献   

6.
枯草芽孢杆菌Bacillus subtilis RF1是课题组前期通过基因工程改造得到的一株核黄素高产菌,为了进一步提高核黄素的产量,需要对该菌株进行进一步的基因工程改造.本研究首先将编码的链丝菌素乙酰基转移酶基因sat克隆到p MA5质粒上,构建具有诺瓦丝菌素Norseothricin(NTC)抗性的重组质粒p MA5-sat,实验证实该重组质粒能够用于B.subtilis RF1的抗性筛选.随后将核黄素合成相关的关键酶葡萄糖-6-磷酸脱氢酶编码基因zwf克隆到重组质粒p MA5-sat上,获得重组质粒p MA5-sat-zwf,并成功构建重组菌B.subtilis RF1/p MA5-sat-zwf.结果显示,重组菌胞内葡萄糖-6-磷酸脱氢酶活力比原始菌提高了近50倍,说明葡萄糖-6-磷酸脱氢酶在重组菌中成功过量表达;根据发酵特性分析,重组菌B.subtilis RF1/p MA5-sat-zwf最终核黄素产量达到12.01 g/L,比原始菌B.subtilis RF1提高了30.3%.综上,本研究构建的新型抗性质粒能够成功运用于核黄素生产菌枯草芽孢杆菌的基因工程改造.  相似文献   

7.
嗜热子囊菌是一种嗜热真菌,可以产生具有很高工业价值的内切葡聚糖酶.本研究成功表达了嗜热子囊菌内切葡聚糖酶Ⅰ基凶,并获得热稳定的重组内切葡聚糖酶.提取嗜热子囊菌光孢变种(Thermoascus aurantiacus var.levisporus)总RNA,通过RT-PCR方法克隆出内切-β-葡聚糖酶eg1基因的成熟肽编码序列.采用基因重组的方法构建该基因的巴斯德毕赤酵母Pichia pastoris分泌型表达载体pPIC9K-eg1,经线性化后采用电穿孔法将其导入毕赤酵母GS115中,大量筛选后获得高效表达内切葡聚糖酶Ⅰ的毕赤酵母工程菌株GpN24.该菌株采用甲醇诱导120 h后,内切葡聚糖酶Ⅰ的活力可达570.7 U/mL,最适温度为55℃,在90℃的条件下保温30 min后仍具有60%的酶活力;最适pH为5.0,在pH 3.0~5.0的条件下酶活力保持稳定.图6表2参16  相似文献   

8.
为获取能表达具有天然活性的杂合抗菌肽的重组酵母菌,确定其最佳表达条件,将构建好的重组酵母表达载体pPICZα-A-CecropinB(1~10)-Magainin2(1~12)(简称pPICZα-A-CBMA)和pPICZα-A-Lfcin-Pro-CecropinB(简称pPICZα-A-LPCB)经SacⅠ线性化处理,分别电转入巴斯德毕赤酵母Pichia pastoris X-33,利用抗生素Zeocin筛选阳性克隆,PCR扩增验证,甲醇诱导表达.采用抑菌圈法初步测定产物活性,优化杂合肽LPCB诱导时间、诱导剂浓度、诱导温度、培养基pH等表达条件,传代检测其质粒稳定性.结果显示,基因工程菌P.pastoris X-33/pPICZα-A-CBMA和P.pastoris X-33/pPICZα-A-LPCB成功构建.表达产物前者抑菌活性微弱,后者抑菌活性良好.杂合肽LPCB的最佳表达条件为:25℃,pH中性培养,每24 h添加0.5%(V/V,φ)甲醇,诱导表达72 h.重组酵母菌pPICZα-A-LPCB在培养超过100代后,未发生质粒丢失.  相似文献   

9.
β-甘露聚糖酶基因在枯草芽孢杆菌中的克隆及表达   总被引:2,自引:0,他引:2  
从Bacillus subtilis JNA 3-10中克隆出β-甘露聚糖酶基因成熟肽链编码序列manA1和含信号肽的β-甘露聚糖酶基因manA2,在B.subtilis 168中克隆表达,分别筛选获得高效分泌表达β-甘露聚糖酶的重组菌株BPM1001(pMA5-manA1/B.subtilis 168)和BPM1002(pMA5-manA2/B.subtilis 168),结果表明菌株BPM1002总酶活力是菌株BPM1001的9.65倍,是原始菌株的13.1倍.在基因manA2下游引入His序列克隆出β-甘露聚糖酶基因manA3,获得枯草芽孢杆菌168重组菌株BPM1003.采用Ni-NTA柱纯化重组菌株BPM1003分泌表达的β-甘露聚糖酶,并研究其酶学性质,该酶促反应的最适pH为6.5,最适温度为65℃,在37℃条件下保存一个月酶活力依然保留有77.8%.5 L发酵罐放大实验结果表明魔芋粉对于产β-甘露聚糖酶具有明显的诱导作用,酶活力最高可达2 748.82 U/mL.图9表3参19  相似文献   

10.
利用PCR方法从解淀粉芽孢杆菌DC 4总DNA中扩增出豆豉溶栓酶 (DFE)成熟肽编码区片段 .测序结果表明 :DFE成熟肽编码区长 82 5bp,编码 2 75个氨基酸残基 ,分子量为 2 7.7× 10 3 ,推导的N’ -端氨基酸序列与豆豉溶栓酶N’ -端氨基酸测序结果完全一致 ,说明克隆到的基因确实是豆豉溶栓酶基因 .同源性分析表明 ,DFE成熟肽编码区的核苷酸和氨基酸序列与日本纳豆激酶的同源性分别为 80 .0 %和 86 .5 % ,这提示豆豉溶栓酶可能是一种新型的溶栓酶 .将表达质粒pET Nde转化E .coliBL2 1(DE3)中 ,IPTG可诱导表达大量的DFE融合蛋白 ,占菌体可溶性蛋白的 4 0 % ,主要以包涵体的形式存在 .图 3表 1参 19  相似文献   

11.
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.  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
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  相似文献   

15.
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.  相似文献   

16.
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.  相似文献   

17.
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.  相似文献   

18.
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.  相似文献   

19.
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.  相似文献   

20.
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.  相似文献   

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