印楝素A无细胞合成体系构建及其合成前体

【目的】印楝素是一种植物源杀虫剂,印楝素A是印楝素的主要活性成分。本研究通过对印楝素A无细胞合成体系制备条件和反应条件的优化,以及印楝素A合成前体的筛选,探讨印楝素A的离体生物合成途径,以为指导构建印楝素异源生物合成平台,实现印楝素的异源生物合成奠定基础。【方法】通过对缓冲液类型、浓度、起始pH、提取时间及料液比进行单因素试验及正交试验优化,构建印楝素A无细胞合成体系;从反应终止试剂及稳定剂、反应温度、反应时间、底物浓度以及辅助因子5个方面优化无细胞合成体系的反应条件。利用印楝素A无细胞合成体系分别以2,3-环氧鲨烯、羊毛甾醇、大戟二烯醇、丁酰鲸鱼醇、nimbin、茄碱苷和脱乙酰茄碱苷为底物,以印楝素A相对产量为评价标准,探讨印楝素A合成前体。【结果】印楝素A无细胞合成体系的构建:以pH7.0的200 mmol·L~(-1) Tris-HCl缓冲液,按1∶20(g·mL~(-1))料液比加入印楝叶片,提取1 h。优化的印楝素A无细胞合成条件为:反应总体积800μL,其中含印楝无细胞提取物300μL(相当于印楝叶0.015 g),100μL 250μmol·L~(-1)的底物(鲨烯)和400μL含有1 mmol·L~(-1) Mg~(2+)、1 mmol·L~(-1) Mn~(2+)、0.1 mmol·L~(-1) ATP、0.1 mmol·L~(-1) NADPH~+和5 mmol·L~(-1)抗坏血酸的缓冲液,在30℃下反应60 min,迅速加入200μL乙酸终止反应。分别以2,3-环氧鲨烯、丁酰鲸鱼醇、大戟二烯醇、nimbin、茄碱苷和脱乙酰茄碱苷为底物,反应完成后,印楝素A含量均有不同程度的增加;羊毛甾醇抑制体系中印楝素A的合成。【结论】羊毛甾醇不是印楝素A的合成前体,2,3-环氧鲨烯和丁酰鲸鱼醇是印楝素A的前体,nimbin、大戟二烯醇、脱乙酰茄碱苷和茄碱苷极可能是印楝素A的合成前体,且茄碱苷位于合成途径较下游的位置。     

快速搅拌法室温浸提印楝油和印楝素

在室温下用间歇式快速搅拌法从印楝种仁中浸提印楝油和印楝素。讨论了料液比以及浸提时间对得率的影响。结果表明:印楝种仁300g,以750mL石油醚做溶剂,在快速搅拌条件下浸提21min时印楝油得率最高;将300g去油后的印楝种仁以900mL甲醇做溶剂,在快速搅拌下浸提21min时印楝素得率最高。由此得出,快速搅拌法浸提印楝油的最佳条件是:溶剂石油醚、料液比1∶2.5(g∶mL,下同)、时间每次7min、浸提3次,出油率30.4%;快速搅拌法浸提印楝素的最佳条件是:溶剂为甲醇、料液比1∶3、时间每次7min、浸提3次,印楝素A的得率为0.438%,印楝素B的得率为0.142%。     

印楝提取物对桑天牛产卵量及卵孵化率的影响

使用一定浓度的印楝提取物 (NeemEC)处理的构树枝条作为天牛的食物 ,研究了其对桑天牛产卵量及卵孵化率的影响。在室温下 ,将成对的天牛放在盖有铁砂网的直径 2 5cm高的塑料桶中进行实验。用含印楝素 50× 1 0 -6g/mL的印楝提取物处理枝条 ,处理组天牛 ( 7对 )在 1 5d试验中 ,其每天对平均产卵量为2 0 0± 1 56个 ,而对照组每天平均产卵量为 1 0 33± 3 33个。经T—检验处理组与对照组之间差异显著。用含印楝素 1 0 0× 1 0 -6g/mL的印楝提取物处理枝条 ,在 1 5d试验中 ,处理组 7对天牛从第 5d开始死亡 ,至第 8d全部死亡 ,按实际成活天数计算 ,其每天每对平均产卵量为 1 0 0± 1 4 1个 ,而对照每天每对平均产卵量为 1 3 73± 2 90个。经T—检验两者之间差异显著。取食含印楝素 50× 1 0 -6g/mL的印楝提取物处理枝条后天牛产的卵需经 1 9d达到其最终孵化率 ,33 33% ;而对照组 9d达到最终孵化率 98 96%。在进行了 2 4d的选择取食试验中含印楝素 1 0 0× 1 0 -6g/mL的印楝提取物处理枝条 ,1 0对对照组天牛每天每对平均取食量为 3 53± 0 38g,而处理组天牛每天每对平均取食量 3 4 2± 0 60 g。经T—检验两者差异不显著。这说明桑天牛不能分辩含印楝提取物的食物 ,即印楝提取物对桑天牛不具拒食性。     

土地利用方式对土壤有机碳和碳库管理指数的影响

土壤碳库管理指数反映土壤管理的科学性.对比研究了干热河谷新银合欢林、苏门答腊金合欢林、大叶相思林、印楝林、荒地和旱耕地土壤有机碳(SOC)、易氧化有机碳(ROC)含量和土壤碳库管理指数.结果表明:6种土地利用方式下SOC和ROC含量范围分别为4.22～5.92 g·kg-1和1.34～2.33 g·kg-1.各利用方式...     

蟑螂水溶性多糖提取、分析及免疫活性研究

采用正交实验设计,以多糖含量为指标,对蟑螂水溶性多糖进行碱液和酶解提取研究;通过离子交换层析、凝胶层析对提取的多糖进行分级纯化,并对所得组分进行纯度鉴定和单糖分析;同时考察蟑螂粗多糖对正常小鼠的免疫活性.结果表明:酶解法提取的多糖含量较高,最佳实验条件是提取温度50℃、酶量200 μg·g-1,提取时间2 h.在此最佳实验条件下所得蟑螂多糖粗品中总糖含量为80 g·kg-1,蛋白含量为423 g·kg-1,氨基酸总量为333.4 g·kg-1;纯化后初步获得一个组分PWSPSC-2,多糖含量458 g·kg-1,蛋白含量392 g·kg-1,单糖组成Xyl:Ara:Glu:Man:Gal:GIcUA:GalUA=1.0:1.7:38.1:14.6:15.6:2.9:1.3;免疫活性实验显示,蟑螂粗多糖可以显著提高小鼠腹腔巨噬细胞吞噬指数、吞噬百分率及血清溶血素,表明蟑螂粗多糖可以明显增强小鼠的非特异性免疫和体液免疫功能.     

桑天牛取食注射印楝素的构树后对其后代死亡率的影响

用注有0.32g和2.0g印楝素的构树枝条喂饲桑天牛一段时间后,能够极显著降低其产卵量,并且其下一代孵化的幼虫成活率降低,多数幼虫在进入木质部取食前因体内的印楝素致其死亡,取食的印楝素越多,死亡越早;注入构树体内的印楝素对接入的正常生长的桑天牛幼虫也有很好的毒杀作用。试验结果为注射印楝素引诱树防治桑天牛的方法提供了理论依据。     

转基因三倍体毛白杨抗盐试验研究

试验设计4～11 g·kg-1 8个NaCl浓度梯度,对转PLD/AtNHXI基因抗盐碱三倍体毛白杨进行组培苗抗盐分化试验,设计2～10 g·kg-19个NaCl浓度梯度进行组培苗抗盐生根试验,设计3～12 g·kg-1 10个NaCl浓度梯度对1年生盆栽苗进行抗盐试验,结果表明:在NaCl浓度达8 g·kg-1时,转基因三倍体毛白杨分化培养37 d后,组培苗的叶色、分化芽数及新稍长度开始受到影响,随着NaCl浓度的增大将抑制组培苗分化生长,甚至枯萎死亡;在含NaCl 7 g·kg-1的生根培养基中,培养20 d后,虽然叶色没有受到盐太大的影响,但生根和新稍的生长都开始受到抑制,根系明显的减少;在含NaCl 7 g·kg-1的土壤条件下,盆栽苗培养32 d后,开始表现出盐害症状.     

配方施肥对印楝人工林产量和生长性状的影响

对元谋干热河谷印楝人工林经3 a施肥试验表明,配方施肥对印楝林分的生长和结实产量均产生显著影响:(1)施肥当年对林分生长未显明显肥效,施肥效应主要表现在施肥后第1年,到施肥后第2年肥效总体呈下降趋势;各生长性状对施肥配方反应的敏感性排序为:冠幅＞株高＞地径＞胸径;以株高和冠幅生长为评价依据,第14处理(300 g氨肥+300 g磷肥+200 g钾肥)为最优施肥组合方案.(2)不同施肥配方处理间以及不同年份之间,印楝的结实产量均存在显著差异;有机肥与P、K肥的组合配方对印楝结实量产生明显增益,其中第1处理(8 kg有机肥+200 g磷肥+200 g钾肥)为增加印楝结实产量的最佳施肥方案;各年份施肥处理间结实产量的排序为:施肥后第1年＞施肥后第2年＞施肥当年.此外,施肥前的林分本底值对林木生长产生极显著影响,在一定程度上干扰了施肥效应.     

金沙江干热河谷印楝适生区域选择初步研究

研究表明:四川凉山金沙江干热河谷地区,限制印楝生长的主要因子是冬春寒害和气温。在本区,寒害随纬度升高而加剧,高于北纬27°10′地区不适宜种植印楝;在低于27°10′的地区,印楝的生长随海拔升高而降低,寒害随海拔升高而加剧。在本区会理县1 200 m海拔内适宜种植,宁南县则必须控制在1 000 m海拔内。     

4种药剂对沙棘绕实蝇成虫的触杀活性

目的]沙棘绕实蝇在我国新疆和内蒙古地区对沙棘果实造成重大危害,如何选取高效、易降解、低残留的农药种类成为当前沙棘绕实蝇成虫防控工作中的迫切课题。[方法]野外采集沙棘绕实蝇蛹,室内待其羽化后,雌、雄分组放置于不同浓度梯度的4种农药条件下进行触杀实验。[结果]通过KT_(50)(median knockdown time)的数据分析结果显示,同种农药同种浓度时,药物对雄性的触杀效果比雌性强,其中苦参碱在浓度为0.005 mg·m L~(-1)时,雄性KT_(50)值为39.21 h,小于雌性KT_(50)值56.13 h,但个别情况下出现对雌性触杀效果比雄性高的情况,如苦参碱在浓度为0.05 mg·m L~(-1)时,雄性KT_(50)值为15.76 h,大于雌性KT_(50)值12.39 h。通过测定4种农药对沙棘绕实蝇成虫的致死中浓度LC_(50)(median lethal dose),得出触杀作用时间为12 h时,LC_(50)分别为:印楝素7.737 mg·m L~(-1)、高效氯氟氰菊酯0.002 mg·m L~(-1)、苦参碱0.042 mg·m L~(-1)、阿维菌素0.008 mg·m L~(-1),其触杀毒力次序为:高效氯氟氰菊酯阿维菌素苦参碱印楝素;触杀作用时间为24 h时,LC_(50)分别为:印楝素6.963 mg·m L~(-1)、高效氯氟氰菊酯0.001 mg·m L~(-1)、苦参碱0.008 mg·m L~(-1)、阿维菌素0.001 mg·m L~(-1),其触杀毒力次序为:阿维菌素高效氯氟氰菊酯苦参碱印楝素;触杀作用时间为48 h时,LC_(50)分别为:印楝素0.891 mg·m L~(-1)、高效氯氟氰菊酯0.001mg·m L~(-1)、苦参碱0.002 mg·m L~(-1)、阿维菌素0.001 mg·m L~(-1),其触杀毒力次序为:阿维菌素=高效氯氟氰菊酯苦参碱印楝素。[结论]阿维菌素和高效氯氟氰菊酯对沙棘绕实成虫的触杀效果较显著,其次为苦参碱,印楝素的触杀效果不显著。     

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.