YK-624产锰过氧化物酶的生产及初步纯化

利用白腐菌PhanerochaetesordidaYK 624(ATCC90872),采用Kirk液体培养基在富氧条件下进行振动培养,可得到锰过氧化物酶(MnP)活性较高和漆酶活性微量的酶液。该酶液通过超滤、透析、二乙氨基乙基琼脂糖凝胶(DEAE sepharoseCL 6B,pharmacia)离子交换柱层析的方法进行初步纯化后,得到比活性很高(1941.54IU/mg)的MnP酶液。漆酶活性和MnP活性比值的变化、SDS PAGE凝胶电泳试验和406nm附近的吸光值变化均表明,经离子交换柱层析后的YK1部分是主要的MnP,电泳试验同时表明纯化后的主要MnP的分子质量约为43kDa。经超滤、透析、离子交换柱层析纯化后得到的酶液YK1可用于进一步地研究及应用。     

限制性内切酶在PCR缓冲液中酶切活性的研究(英文)

选取6 种常用的限制性内切酶(Acc I、Ban II、EcoR I、Hind III、Sac I、Sca I)用于检测其直接在PCR 缓冲液中完全酶切DNA 的活性。结果表明:在PCR 缓冲液中,需要加入MgCl2作为激活因子的前提条件下,除了EcoR I 表现出星状活性外,其他酶均能完全酶切无甲基化λDNA,其结果与在酶生产厂商推荐的反应缓冲液中进行的酶切反应结果一致;在完成PCR 扩增的PCR 缓冲液中,所有酶(包括RcoR I)均能功能正常地对PCR 产物完全酶切,表明可在PCR 扩增完毕后的反应体系中直接加入限制性内切酶进行酶切反应,无需对PCR 产物进行预先纯化;镁离子浓度在2.5 mmol·L-1至10 mmol·L-1范围内对PCR 缓冲液中的限制性内切酶的活性无显著影响。上述方法可用于限制性片段长度多态(RFLP)以及单链构像多态(SSCP)研究中,但能否用于分子克隆有待进一步实验。图3 表1 参7。     

黄绿木霉固定化生产纤维素酶及酶学特性的研究

利用海藻酸钙凝胶包埋法制备固定化细胞黄绿木霉,结果表明:海藻酸钠质量浓度50 g/L,培养基初始pH值4.0~4.5,以滤纸浆为培养基碳源培养的小球的稳定性好。发酵产酶培养时以滤纸浆为碳源,海藻酸钠质量浓度为50 g/L,CaC l2质量浓度控制在20 g/L,培养基初始pH值选择为4.5,产酶效果好、酶活力高、保持时间长。添加表面活性剂Tw in-80后,产酶能力可进一步提高。通过(NH4)2SO4分级沉淀、Sephadex G-100分子筛层析和DEAE Sephadex A-50离子交换层析等步骤,分离纯化出黄绿木霉纤维素酶系中达到电泳纯的3种内切葡聚糖酶(EGⅠ、EGⅡ、EGⅢ)和2种β-葡萄糖苷酶(BGⅠ、BGⅡ)。通过SDS-PAGE和IEF电泳测得5个酶组分的相对分子质量(Mr)分别为62 300、71 900、52 600、85 300和78 300,等电点分别为5.4、4.8、5.0、5.6和5.8。     

漆树酶的固定化——Ⅱ对氨基苯砜乙基(ABSE-)交联琼脂固定化漆树酶的研究

漆树酶(EC.1.10.3.2)共价偶联在对氨基苯砜乙基(ABSE-)交联琼脂上。研究了固定漆树酶的条件,比较了琼脂与琼脂糖两种固定化酶载体。固定化漆树酶的活力达23光密度变化值/分·克(干),活力回收达37％。找出了固定化漆树酶的最适温度和最适pH,测定了它的米氏常数,热稳定性和重复使用性,并且与天然漆树酶进行了比较。探讨了残余重氮盐对固定化酶的影响。固定化漆树酶能重复使用二十次以上,其热稳定性也优于天然漆树酶。     

思茅松等位酶实验方法及改进

以思茅松种子的胚乳为实验材料，探索了思茅松等位酶的实验方法。采用聚丙烯酰胺垂直板凝胶电泳与水平切片淀粉凝胶电泳相结合的方法，获得所试验的13种等位酶中AAT，ADH，EST，GDH，G6PD，MDH，PGD-6，PGM和SKD等9种电泳效果良好的等位酶酶谱。通过两种电泳方法的有机结合，以及对制样及上样方法的改进，大大地提高了实验效率。本研究得到的等位酶酶谱式样，为下一步思茅松种群遗传分析提供了资料。     

思茅松等位酶实验方法及改进

以思茅松种子的胚乳为实验材料,探索了思茅松等位酶的实验方法.采用聚丙烯酰胺垂直板凝胶电泳与水平切片淀粉凝胶电泳相结合的方法,获得所试验的13种等位酶中AAT,ADH,EST,GDH,G6PD,MDH,PGD6,PGM和SKD等9种电泳效果良好的等位酶酶谱.通过两种电泳方法的有机结合,以及对制样及上样方法的改进,大大地提高了实验效率.本研究得到的等位酶酶谱式样,为下一步思茅松种群遗传分析提供了资料.     

漆酶高产菌株筛选及漆酶基因片段的克隆

研究利用不同底物浓度、不同培养时间、不同培养基为优化因素,对漆酶产生菌株筛选条件进行了初步研究,从广泛4株食用真菌中筛选具有高产漆酶特性的真菌,双孢菇2796,并以双孢菇2796为供体菌,提取菌株RNA,运用RT-PCR方法对漆酶基因进行扩增,克隆后得到长度为160bp的漆酶基因片段。     

过氧化物酶同工酶与银杏性别及年龄的关系

采用聚丙烯酰胺凝胶电泳的方法对已知性别的银杏成年大树（树龄４０年以上）、在形态上尚未分化出雌雄的实生苗（苗龄２～３年）、实生幼苗（苗龄４０天）以及雌雄树叶诱导的愈伤组织进行过氧化物酶同工酶的测定。经重复实验，发现成年树雌雄株的酶谱存在着差异，雌株较雄株有较多的酶带，且有两条酶带的电泳迁移率相同，但活性不同。实生苗和实生幼苗酶带比成树的明显减少，且在不同株中的活性趋于相同。而对已知性别成年树叶片诱导的愈伤组织进行检测，其酶谱与成树、实生苗和实生幼苗的明显不同，但均出现了雌雄成树共同具有一条酶带，且活性极强。但过氧化物酶同工酶图谱能否作为鉴定银杏植株早期性别鉴定的生理生化指标，尚需进一步探索和研究。     

PEG6000和BSA促进外切葡聚糖酶的水解机制（英文）

表面活性剂及牛血清白蛋白已被证实能促进纤维素酶的水解,但其促进水解的机制仍不清楚。对表面活性剂PEG6000及牛血清白蛋白BSA影响关键纤维素酶组分(内切葡聚糖酶Ⅱ,外切葡聚糖酶Ⅰ和β-葡萄糖苷酶)水解微晶纤维素的能力进行了研究。结果表明,PEG6000和BSA可将微晶纤维素的水解得率从42.7%分别提高到61.6%和52.2%。内切葡聚糖酶Ⅱ和β-葡萄糖苷酶的水解能力并不能被表面活性剂PEG6000和BSA所促进。吸附试验证实PEG6000能减少内切葡聚糖酶Ⅱ和外切葡聚糖酶Ⅰ对纤维素的吸附。动力学研究表明,PEG6000和BSA是外切葡聚糖酶Ⅰ的激活剂,但并不是内切葡聚糖酶Ⅱ和β-葡萄糖苷酶的激活剂。因此,PEG6000和BSA对外切葡聚糖酶Ⅰ的激活作用是其促进纤维素酶水解的关键机制。     

黄蓝状菌一种几丁质酶的纯化、性质及抗菌活性

黄蓝状菌Talaromyces flavus在SMCS液体培养基中置于恒温摇床(200r/min,27．5℃)上培养13d，诱导产生大量的几丁质酶。培养滤液经硫酸铵分级沉淀，DEAE-Sepharose Fast Flow阴离子交换层析、Phenyl-Sepharose Fast Flow疏水层析、Sephacvl S—100分子筛层析得到了凝肢电泳(SDS-PAGE)谱带单一的几丁质酶，其分子量为41kD。其最适反应温度为50℃，最适pH值是5。Mn^2 对其有明显的激活作用，Cu^2 、Fe^2 对其有强烈的抑制作用。抗菌活性显示，其对供试病原菌具有明显的抑菌作用。     

Browsing Damage on Pine (Pinus sylvestris and P. contorta) by a migrating moose (Alces alces) Population in Winter: Relation to Habitat Composition and Road Barriers

In Scandinavia, moose (Alces alces L.) sometimes cause severe browsing damage to economically-important pine. Moose-vehicle accidents have spurred construction of fences along roads, and these may interfere with moose migration between summer and winter ranges, or the road alone may be a barrier. If this happens and moose build up along roads, landowners may suffer economically. Therefore, this study investigated whether roads, fences or other factors influence the use of young pine stands by moose. Eighty stands along roads in northern Sweden were evaluated in which individually-browsed branches were counted on 9972 pines. Moose browsing was not significantly related to birch (Betula pendula Roth, B. pubescens Ehrh.) density, nor did it differ between pines (Pinus contorta Douglas or P. sylvestris L.). However, increased pine density, site productivity and proximity to a highway were associated with increased browsing. Further large-scale studies are needed to understand moose habitat selection and the effects of roads.     

Effects of Root Freezing on the Physiology and Growth of Picea glauca,Picea mariana and Pinus banksiana Seedlings Under Different Soil Moisture Regimes

The root systems of 2-yr-old Picea glauca, Picea mariana and Pinus banksiana seedlings were submitted to various frost temperatures during an artificial frost to induce different levels of root damage. Frost-damaged and control seedlings were placed in a greenhouse under high and low soil moisture regimes. Seedling growth and physiology were evaluated periodically. Seedling survival was reduced when root damage reached levels of 60-80%. Root systems of all three species showed partial to total recovery by the end of the experiment. In general, root freezing damage caused reductions in seedling growth, with these reductions becoming less significant over time. Root damage had little to no effect on black spruce and jack pine seedling physiology, while white spruce CO 2 uptake decreased with increasing root damage. Shoot nitrogen content of all three species decreased slightly with increasing root damage.     

Stand Structure in Pine,Spruce and Deciduous Forests: A Comparison Between Finnish and Russian Karelia

This study investigated the stand structure in pine, spruce and deciduous forests in the border district of Finland and Russia. A total of 46 mature forest stands was selected as pairs, the members of each pair being as similar as possible with respect to their forest site type, age, moisture and topography. The stands were then compared between the two countries by means of basal areas and number of stems. The proportions of dominating tree species were 2-12% lower, and correspondingly the proportions of secondary tree species higher, in Russian forests. The density of the forest stock was also higher in each forest type in Russia. The forests in the two countries differed most radically in terms of the abundance of dead trees. The amount was two to four times higher in Russian deciduous and spruce forests, and in pine forests the difference was 10-fold. The stand structures indicated that Russian coniferous stands, in particular, were more heterogeneous than intensively managed pine and spruce stands in Finland.     

Growth and Terminal Bud Formation in Picea abies Seedlings Grown with Alternating Diurnal Temperature and Different Light Qualities

The root collar diameter and the height:diameter ratio are of particular importance in container-grown seedlings where a high density in the containers may produce spindly seedlings. Temperature regimes and light quality are known to affect plant growth. The aim of this study was to identify responses in Picea abies (L.) Karst. seedlings grown with light providing different red:far-red ratios and under temperature regimes with alternating day (DT) and night temperature (NT) from negative (DT < NT) to positive (DT > NT) difference (DIF) between DT and NT. Experiments were conducted in controlled environment chambers and in a daylight phytotron. Only limited thermoperiodic responses appeared in P . abies seedlings with respect to seedling height and dry weight accumulation. The formation of terminal buds, however, was clearly delayed in seedlings grown at negative DIF. The results indicate a requirement for day extension light that is high in far-red, to prevent terminal bud formation under natural short-day conditions. An extended study should be conducted to clarify the minimum level of light intensity and the optimal light quality needed to prevent terminal bud formation under natural short-day conditions.     

CAD与建筑设计的关系

本文分析了CAD在设计中引起的正面和负面影响,并进行了系统的阐述,从而使设计者在应用中能够保持客观的态度。     

Natural Regeneration in Clear-cuts: Effects of Scarification,Slash Removal and Clear-cut Age

The effects of soil scarification (mounding), slash removal and clear-cut age on the natural regeneration in clear-cuts was evaluated using data from four sites in southern Sweden. The treatments were carried out during a good seed and establishment year for birch ( Betula pubescens Ehrh. and B. pendula Roth). Scarification had the strongest positive effect on the density of naturally regenerated seedlings, especially in birch, but also in pine ( Pinus sylvestris L.) and spruce [ Picea abies (L.) Karst]. Slash removal had a positive effect on birch density. No statistically significant effect of clear-cut age was found. In addition, no statistically significant interactions between clear-cut age and scarification or slash removal were found. The ingrowth of field vegetation was the fastest in areas that were not scarified, less rapid in areas scarified on old clear-cuts, and the slowest after scarification in fresh clear-cuts. In conclusion, it may be possible to control the density of birch during a good establishment year for birch. If birch is desired, the best combination of treatments is to remove the slash and scarify; otherwise, these treatments should be avoided.     

Risk Perception Among Non-industrial Private Forest Owners

Based on an enquiry, risk perception among non-industrial private forest owners is described in relation to climate change and forestry hazards. Of the respondents, 11% took action to remedy the effects of climate change. Out of a given set, hazards were ranked according to each respondent's experience of recent substantial financial loss to the estate and in relation to his or her willingness to make investments aimed at risk reduction. For each hazard, the respondent assessed the risk in four classes ranging from very high to negligible risk. Six hazards were considered most problematic in all three aspects: browsing damage, falling timber prices, damage by wind, spruce bark beetle, root rot and pine weevil. A majority of the respondents claimed to take action to reduce the risk associated with at least one hazard, while 35% did not know whether they did. Excluding climate change, the need for decision support was the largest in relation to damage by wind owing to a combination of perceived high risk and a high level of ignorance in relation to whether risk-reducing measures were taken.     

Interactions in decomposition and N mineralization between tropical legume residue components

In situ produced plant residues contain a mixture of different plant components of varying quality. To assess synergistic or antagonistic interactions occurring during the decomposition and mineralization of such mixtures, individual plant parts (stems, leaves, leaf litter and roots) or the mixture of stems, leaves and leaf litter of the agroforestry species pigeonpea (Cajanus cajan) or of crop residues of peanut (Arachis hypogaea) or of the weed hairy indigo (Indigofera hirsuta) were incubated in pots for 19 weeks. Periodically, remaining plant residues were sieved out (>2 mm), weighed and N content as well as soil mineral N determined. Above- and below-ground residues of peanut decomposed fastest and showed the largest N release in agreement with their high N concentration and low-acid detergent fibre (ADF) : N ratio. Hairy indigo was hypothesized to be of lower quality than pigeonpea because of its high-polyphenol content. However, it decomposed faster than pigeonpea, largely because of the higher N and lower lignin concentration of its components. Ranking of individual plant components for N mineralization resulted in the following pattern, leaves > leaf litter > roots > stems. In mixtures of the different plant components a similar species order in decomposition was obtained, e.g. peanut > hairy indigo > pigeonpea. The amount of N released from the mixture was dominated by stem material that comprised 46–61% of the mixture. The interactions in mixtures were relatively small for peanut (generally high-quality components) as well as for pigeonpea (low proportion of high-quality components, i.e. N rich leaf material). However, a positive interaction occurred during later stages of N mineralization in the mixture of hairy indigo as it had a significant proportion of N rich components and absence of highly reactive polyphenols. Thus, for plants with low to intermediate chemical quality attributes, manipulating plant composition (e.g. by varying harvest age, affecting stem and leaf proportions) will be important to obtain significant interactions during decomposition when its components are mixed.     

Pasture production and tree growth in a young pine plantation fertilized with inorganic fertilizers and milk sewage in northwestern Spain

The use of organic waste materials such as milk sewage as an organic fertilizer could have the dual advantages of organic-waste disposal and reduced dependence on inorganic fertilizers. The effects of fertilization with (1) conventional mineral fertilization, (2) milk sewage sludge at 40 kg N ha⁻¹ target rate and (3) no fertilization on pasture production and tree growth were examined in an experiment consisting of two pasture mixtures under a one-year-old Pinus radiata plantation with a density of 2500 trees ha⁻¹. The two pasture mixtures were: (1) Dactylis glomerata L. var. saborto (25 kg ha⁻¹) + Trifolium repens L. group Ladino (4 kg ha⁻¹) + Trifolium pratense L. var. Marino (1 kg ha⁻¹); (2) Lolium perenne L. var. Tove (25 kg ha⁻¹) + Trifolium repens L. group Ladino (4 kg ha⁻¹) + Trifolium pratense L. var. Marino (1 kg ha⁻¹). The experiment began in the spring of 1995 using a randomized block design with three replicates in Castro Riberas de Lea (Lugo, Galicia, north-western Spain). Plot size was 12 × 8 m², with a 1 m buffer strip between plots. Two-year data showed that fertilization with either material had a positive effect on pasture production, with no significant difference between the two fertilization treatments. Tree growth in the milk sewage sludge plot was significantly higher than in the control plots. Inorganic fertilization increased pasture production, but affected tree growth negatively. The results show that milk sewage sludge could be used as a fertilizer in silvo-pastoral systems. This revised version was published online in June 2006 with corrections to the Cover Date.