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细菌复制相关解旋酶与引物酶相互作用研究
No full text解旋酶是一类在生物体内普遍存在的蛋白酶，它在DNA复制、修复、重组、转录、维持端粒稳定、RNA加工翻译和降解方面具有非常重要的作用。研究表明DnaB解旋酶在细菌DNA复制过程中具有重要作用，其水解ATP获取能量催化双链核酸解链。在复制起始过程中，解旋酶需要和其他蛋白相互作用构成引发体才能共同完成DNA复制。解旋酶DnaB与引物酶DnaG是引发体的主体，二者产生相互作用可调控彼此的生物活性，DnaG能够促进解旋酶的ATP水解能力和与DNA的结合能力，从而激活解链功能加快复制进程，而DnaB可激活DnaG合成RNA的能力，提高复制效率。已有的晶体结构表明，解旋酶形成环状六聚体；在结合引物酶C端结构域时，仍保持环状六聚体结构；而当解旋酶结合ssDNA时，解旋酶/ssDNA复合物则形成螺旋纤维状结构。但解旋酶在结合了ssDNA后如何和引物酶相互作用仍然不清楚。为了研究DnaB解旋酶与DnaG引物酶之间的相互作用关系，探讨解旋酶与引物酶在DNA复制过程中的作用机制，本文重组表达了嗜热脂肪芽孢杆菌（Geobacillus stearothermophilus）来源的DnaB和DnaG蛋白，通过亲和层析、离子交换层析和凝胶过滤层析纯化得到高纯度的重组DnaB和DnaG蛋白。在此基础上，利用凝胶过滤层析分离制备了DnaB/DNA/DnaG复合物，SDS-PAGE和Native-PAGE检测证明复合物中存在DnaB、DnaG和DNA组分，动态光散射分析结果表明复合物是单分散的，均一的，可用于后续实验。通过孔雀石绿定磷法检测复合物中解旋酶ATP酶活性，发现解旋酶结合DNA或引物酶后ATP酶水解活性提高了一倍。将DnaB/DNA/DnaG（引物酶全长蛋白或其C端结构域HBD）复合物进行结晶筛选和优化，得到了可用于衍射的晶体，结构解析发现复合物中无核酸存在，全长引物酶也发生了降解，整体结构为DnaB/HBD。分析可能是由于池液的环境不利于复合物晶体生长，或是因为DnaG的结合致使DnaB与DNA亲和力下降从而不易形成稳定的复合物。DNA片段的长度及类型也有可能会影响到DnaB对DNA的亲和力，从而影响复合物的稳定性和结晶。后续可从这些方面对复合物稳定存在和结晶进行深入的研究，以期获得复合物的晶体结构。冷冻电镜能在维持复合物稳定的情况下成像，遂尝试利用冷冻电镜解析复合物的结构。在复合物样品的电镜观察中，发现整个颗粒并不是平面六次对称的，而是具有一定的旋转对称特征，猜测解旋酶结合了DNA由平面六聚体变成螺旋状结构，在整个颗粒的外层有一处或者两处突出的密度，推测可能为结合解旋酶的引物酶。目前，由于复合物三维重构分辨率较低，我们还未获得其精细结构。另外，本论文还对引物酶与含特异起始位点的ssDNA结合模式进行了研究。重组表达了枯草芽孢杆菌（Bacillus subtilis）引物酶DnaG，DnaG全长蛋白结晶得到的晶体经X-射线衍射技术只解析了其RPD结构域结构。从蛋白易降解和稳定性差的角度分析了全长引物酶不易结晶的原因。在已有的RPD/ssDNA和RPD/NTP结构研究基础上，利用定点突变实验提出了RPD/ssDNA/NTP结合模型。该模型详细描述了在引物酶起始引物合成阶段模板DNA的结合区域以及DNA序列上特异位点结合的情况
青霉菌CIB-411次级代谢产物及其生物活性研究
No full text青霉属于子囊菌亚门不整囊菌纲散囊菌目散囊菌科青霉属；因其蓝绿色孢子而得名，目前已报道230个种。青霉常见于柑桔类水果、肉食、蔬菜及皮革上，在海洋、陆生植物内生菌中也有发现青霉菌的存在。由于弗莱明从特异青霉菌(P. notatum)发现具有抗菌效果的青霉素，从而改变了人类与传染病之间生死搏斗的历史，因此青霉菌代谢产物受到越来越多的关注与研究。本论文对青霉菌CIB-411的次级代谢产物进行了系统研究，本论文由三章组成。第一章报道了青霉菌CIB-411的分离纯化与鉴定。利用涂布、划线等微生物分离纯化方法，从大曲中分离得到23株真菌，通过扩散法对分离得到的菌株进行了抗菌活性研究。活性结果表明菌株CIB-411的固态发酵提取物具有显著的抗真菌活性。利用ITS序列分析方法对真菌CIB-411进行鉴定，通过与Genbank数据库进行同源性比对，再结合形态学分析结果，最后将真菌CIB-411鉴定为青霉菌，命名为青霉菌CIB-411。第二章报道青霉菌CIB-411的次级代谢产物的研究。通过簿层层析、柱层析、凝胶柱层析以及高效液相色谱法等色谱分离技术和手段，从青霉菌CIB-411大米固态发酵物中分离得到25个化合物（1-25），综合利用NMR、HR-ESI-MS、UV、IR等波谱技术完成了化合物1-25的结构鉴定，分别为：Penazaphilones A-F（1-6），Sclerotioramine (7)，compound 8（8），Isochromophilone VI (9），Isochromophilone IX (10)，4-O-Demethylsilvaticol（11），Acids II（12），索青霉素（13），N-demethylsambutoxin（14），Carnemycin B （15），compound 16（16），Stromemycin（17），（22E,24R)-麦角甾醇-5, 7,22-三烯-3β-醇（18），Cryptosporioptide A（19），Sambutoxin A-E（20-24），Pestalasin E（25）；其中包括11个新的化合物，分别为Penazaphilones A-F（1-6）和sambutoxin A-E（20-24）。活性测试表明，化合物19对金黄色葡萄球菌（SA多重耐药菌）具有较好的抑制活性，抑菌圈直径为12.94mm（纸片直径为5.68mm）；而化合物15对绿脓杆菌（PA），鲍曼不动杆菌（AB）以及金黄色葡萄球菌(SA)均有一定的抑制活性；抗肿瘤活性结果表明，化合物20对肿瘤细胞株A549，B16，HEPG2和SW620均有72%以上的抑制率。第三章概述了Azaphilones 类化合物在结构类型及生物活性方面的研究进展
浓香型白酒发酵过程中微生物群落动态变化研究
No full text中国浓香型白酒是在封闭窖池内由固定的微生物群落代谢发酵而生成。其生产工艺通常采用经典的自然固态发酵方式，以糟醅为发酵基质，窖泥中的微生物、曲药中的微生物和环境中的微生物通过分解糟醅中的有机物质发生复杂的物质能量代谢，生成丰富的与白酒质量密切相关的代谢物质。因此，微生物群落在浓香型白酒整个发酵过程中起到了至关重要的作用。然而，到目前为止窖池内复杂的微生物群落结构和发酵机制依然没有很清楚的为人所知。本研究采用模拟发酵的方法，对发酵第1，10，23，34，48，59和70天的发酵糟醅样品在Miseq测序平台进行16S rRNA和ITS扩增子测序，同时利用高效液相色谱法（HPLC）和气相色谱法（GC）对其理化因子的测定，从而获得发酵过程中不同发酵阶段微生物群落结构及动态演变情况，通过分析微生物群落与关键呈香物质的相关性，进一步可以得出与白酒风味特征有紧密相关性的微生物群落。在分析了微生物群落的基础上，本研究选取了第23，34，48，70天的发酵糟醅样品在Illumina Hiseq平台继续进行宏基因组DNA的测序，对其功能基因结构和代谢特征进行分析，从基因水平上深入了解发酵过程中微生物群落的动态变化。在发酵前期（1-23天），由大分子淀粉、纤维素等大分子糖类物质降解的葡萄糖含量急剧增加，原核微生物群落多样性快速下降，乳酸菌属（Lactobacillaceae）逐渐成为原核群落的绝对优势菌群。然而，真核微生物群落多样性在此过程中却有显著增长，嗜热子囊菌(Thermoascus), 曲霉属（Aspergillus）,根霉属（Rhizopus）和未鉴定的酵母菌目（Saccharomycetales）为主要的真菌优势菌群。在发酵中期（23-48天），葡萄糖含量急剧下降，乙醇和乳酸含量有显著增长。原核群落几乎由乳酸菌属（Lactobacillaceae）构成，而真核群落主要由嗜热子囊菌（Thermoascus）, 裸胞壳属（Emericella）和曲霉属（Aspergillus）组成。在发酵后期（48-70天），酯类物质开始产生并有显著的增长，尤其己酸乙酯含量增长比较明显。整个发酵过程经历了三个不同的发酵阶段：糖化阶段，糖酵解阶段和酯化阶段。酵母菌目（Saccharomycetales）,红曲霉属（Monascus）和根霉属（Rhizopus）与葡萄糖含量呈明显的正相关性，说明其在大分子物种的糖化阶段起了重要作用。乳酸菌科（Lactobacillaceae）,芽孢杆菌纲（Bacilli）,孢盘菌属（Botryotinia）,曲霉属（Aspergillus）,未鉴定的格孢菌目（Pleosporales）和未鉴定的煤炱目（Capnodiales）与有机酸和酯类物质具有明显的相性，说明它们在有机酸和酯类的生成过程中起了关键作用。另外，有四株菌裸胞壳属（Emericella）, 乳牛杆菌属（Suillus）,被孢霉属（Mortierella）和孢盘菌属（Botryotinia）第一次发现存在于糟醅微生物中。通过与四个不同的数据库比对，结果发现，比对到KEGG数据库中的注释基因中，参与新陈代谢的基因注释数量最多。有6类与糖类代谢相关的蛋白酶类基因注释到CAZy 数据库，数量较高的前4位依次为：glycoside hydrolases（糖苷水解酶），carbohydrate-binding modules（碳水化合物结合模块），glycosyl transferases（糖基转移酶）。而比对到eggNOG数据库的直系同源基因簇中，大部分基因簇功能未知。除了注释基因，本研究还注释到了3条第三级水平上的参与环境信息处理的信号转导通络和参与遗传信息处理的翻译通路，分别为磷脂酰肌醇3-激酶信号传导通路（PI3K-Akt signaling pathway），磷脂酰肌醇信号系统通路（Phosphatidylinositol signaling system）和氨酰tRNA合成通路（Aminoacyl-tRNA biosynthesis）。这三条通路主要功能是抵御外来环境中不利于微生物生长的外界刺激，维持窖池内微生态的稳定与代谢。这与浓香型白酒发酵过程中特殊的极端发酵条件有着直接关系。本研究旨在为浓香型白酒的生产实践提供理论上的指导，有助于进一步更加准确地控制浓香型白酒的发酵过程，优化和改善生产工艺
小麦热激蛋白转录因子家族全基因组鉴定、分类和比较分析
No full text热激蛋白转录因子(heat shock transcription factors,HSFs)在植物发育和抵御环境胁迫中发挥重要作用。本研究利用生物信息学方法,在小麦中预测出39个HSF基因,其中29个属于A类,6个属于B类,4个属于C类。然后,我们系统地对这39个TaHSF的染色体位置,基因结构,保守蛋白基序,进化关系及调控网络进行了分析。结果发现,除7A,7B,7D外,其他染色体均有分布FaHSF。与拟南芥,玉米和水稻相比,小麦HSF与短柄草HSF具有更高的同源性。基于同源性方法,TaHSFs与其他小麦基因的调控网络图预测结果显示,6个TaHSF与拟南芥具有同源性,它们与对应313个小麦基因具有调控关系。并且,通过利用已有的RNA-seq数据,对这些TaHSF在小麦生长发育和干旱和高温胁迫下的表达进行了预测。结果发现,TaHSFs在籽粒,叶片,根,穗和茎中的表达,以及干旱和高温胁迫下的表达是不同的。本研究首次报道了小麦中HSF基因家族的基因结构,进化特征和表达情况,这为进一步研究TaHSF在小麦中的功能提供了一定地基础
Silver-catalyzed efficient synthesis of enaminones from propargyl alcohols and amines
No full textEnaminones are used as the key intermediates to construct heterocyclic compounds with various bioactivities. In this study, a simple and efficient approach for the synthesis of enaminones via amination of propargyl alcohols was developed. Under the catalysis of Ag2CO3, the reaction proceeded smoothly to afford the desired products in good yields. Preliminary mechanism experiments showed that Ag2CO3 played an essential role in the procedure of the reaction. (C) 2017 Published by Elsevier Ltd
Differential responses of body growth to artificial warming between parasitoids and hosts and the consequences for plant seed damage
No full textTemperature increase may disrupt trophic interactions by differentially changing body growth of the species involved. In this study, we tested whether the response of body growth to artificial warming (similar to 2.2 degrees C) of a solitary koinobiont endo-parasitoid wasp (Pteromalus albipennis, Hymenoptera: Pteromalidae) differed from its main host tephritid fly (Tephritis femoralis, Diptera: Tephritidae; pre-dispersal seed predator), and whether the plant seed damage caused by wasp-parasitized and unparasitized maggots (larval flies) were altered by warming. In contrast to the significant and season-dependent effects of warming on body growth of the host tephritid fly reported in one of our previous studies, the effect of artificial warming on body growth was non-significant on the studied wasp. Moreover, the warming effect on seed damage due to unparasitized maggots was significant and varied with season, but the damage by parasitized maggots was not altered by warming. Distinct responses of body growth to warming between parasitoids studied here and hosts assessed in a previous study indicate that temperature increase may differentially affect life history traits of animals along food chains, which is likely to affect trophic interactions
Ecosystem carbon stock across a chronosequence of spruce plantations established on cutovers of a high-elevation region
No full textOCT 24-28, 2015This study quantified the above- and belowground carbon (C) stocks across a chronosequence of spruce (Picea asperata) plantations established on cutovers and explored the turning point after which the increase in biomass C slowed or biomass C decreased for guiding forest management. We assessed above- and belowground plant biomass stocks at 11 sites in three regions, representing 12- to 46-year-old spruce plantations established on clear-cut areas in the eastern Tibetan Plateau, China. Biomass and C stocks of trees, understory vegetation, and forest floor litter were determined from plot-level inventories and destructive sampling. Fine root (< 2 mm) biomass and mineral soil organic C (SOC) stock were estimated from soil cores. Tree biomass was quantified using allometric equations based on diameter at breast height (DBH) and height (H). Plant biomass C stocks in spruce plantations rapidly increased from 12 to 20 years at a rate of 7.8 Mg C ha(-1) year(-1), but decreased from 25 to 46 years at a rate of 0.79 Mg C ha(-1) year(-1). SOC stocks in spruce plantations gradually decreased from 12 to 46 years at a rate of 4.4 Mg C ha(-1) year(-1). Total C stock in the ecosystem remained unchanged for the first 20 years after the planting of spruce on cutovers, because the buildup of C stock in spruce biomass during the first 20 years was offset by the decrease in SOC. From 21 to 46 years after the reforestation, ecosystem C stock even decreased at a rate of 5.2 Mg C ha(-1) year(-1). The contribution of the understory vegetation, forest floor litter, and fine root to ecosystem C stock was low (< 5.0 %) in the spruce plantations. Ecosystem C stock in the spruce forest established on the cutover in the eastern Tibetan Plateau was related to stand age. During the first 20 years, this ecosystem was C neutral. However, aged (20-46 years) spruce plantation ecosystem can be a C source if no management was implemented to revitalize tree growth, promote understory vegetation, and enhance SOC accumulation
The coupling effects of water deficit and nitrogen supply on photosynthesis, WUE, and stable isotope composition in Picea asperata
No full textWater stress and nitrogen (N) availability are the two main factors limiting plant growth, and the two constrains can interact in intricate ways. Moreover, atmospheric N depositions are altering the availability of these limiting factors in many terrestrial ecosystems. Here, we studied the combined effects of different soil water availability and N supply on photosynthesis and water-use efficiency (WUE) in Picea asperata seedlings cultured in pots, using gas exchange, and stable carbon and nitrogen isotope composition (delta C-13 and delta N-15). Photosynthesis under light saturation (A(sat)) and stomatal conductance (g(s)) of P. asperata decreased as the soil moisture gradually diminished. Under severe water-stress condition, N addition decreased the Asat and gs, whereas the positive effects were observed in moderate water-stress and well-watered conditions. The effect of N addition on the intrinsic WUE (WUEi) deduced from gas exchange was associated with soil water availability, whereas long-term WUE evaluated by leaf delta C-13 only affected by soil water availability, and it would be elevated with soil moisture gradually diminished. Water deficit would restrict the uptake and further transport of N to the aboveground parts of P. asperata, and then increasing delta N-15. Therefore, delta N-15 in plant tissues may reflect changes in N allocation within plants. These results indicate that the effect of N enrichment on photosynthesis in P. asperata is largely, if not entirely, dependent on the severity of water stress, and P. asperata would be more sensitive to increasing N enrichment under low soil water availability than under high soil moisture
The Synergistic Responses of Different Photoprotective Pathways in Dwarf Bamboo (Fargesia rufa) to Drought and Subsequent Rewatering
No full textDwarf bamboo-dominated forests are often subjected to temporary periods of drought due to rising air temperature and decreasing rainfall. Nevertheless, the relationship among CO2 assimilation, photoprotective pathways and metabolism of reactive oxygen species (ROS) remains unexplored in bamboo species. Changes in leaf gas exchange, chlorophyll fluorescence, energy partitioning, antioxidative system and compounds related to ROS metabolism in Fargesia rufa plants subjected to drought and subsequent rewatering were analyzed. Drought resulted in a reversible inhibition of photochemistry, particularly net CO2 assimilation, and lipid peroxidation due to ROS accumulation. Meanwhile, photoprotective pathways, including the water-water cycle (especially for moderate drought), and adjustment in antenna pigments, thermal dissipation and antioxidative defense capacity at organelle levels (especially for severe drought), were up-regulated at the stress phase. Conversely, photorespiration was down-regulated after drought stress. As a result, rewatering restored most of the photochemical activity under drought, especially moderate drought. Moreover, thermal dissipation under severe drought was still operated for avoiding high ROS levels after rewatering. Therefore, the synergistic function of these photoprotective pathways except photorespiration can protect the photosynthetic apparatus from oxidative damage in response to varying intensities of drought stress when CO2 assimilation is restricted. This is helpful for the gradual recovery of photosynthetic capacity after rewatering. Thus, F. rufa plants can withstand drought and is capable of survival in such environment
Household Livelihood Strategies and Implication for Poverty Reduction in Rural Areas of Central Nepal
No full textUnderstanding household livelihood strategies is pivotal to minimize rural poverty in the least developed countries like Nepal. This study is an attempt to assess livelihood strategies pursued by rural households, investigate the most remunerative strategy, and identify the factors that influence a household's choice of better strategies in rural Nepal. Primary data collected in 453 households from three villages of central Nepal are analyzed quantitatively within a sustainable livelihood framework. This study categorized households into five main livelihood strategy groups. The results showed that the majority (61%) of the households diversified their income to non-farm sources. Livelihood diversification to business/enterprise strategies adopted by 16% of the households is the most remunerative strategy followed by commercial farming that includes 13% of the sample and are more relevant to poverty reduction. Land holding, education, agriculture and skill training, access to credit, and proximity to the road and market center are the major influencing factors on the adoption of higher returning livelihood strategies. Stimulating poor households to follow market-oriented farm and non-farm activities by improving access to education, vocational training, rural credit, and rural infrastructures is momentous for reducing poverty in the rural areas of central Nepal