一种适合油桐种仁蛋白质分离的双向电泳技术体系

为奠定油桐蛋白质组学研究的基础,针对影响油桐种仁蛋白质分离效果的几个重要因素进行单因素优化实验,建立了油桐种仁蛋白质组分离效果良好的双向电泳技术平台。实验结果显示:采用TCA-丙酮结合酚抽法提取的油桐种仁蛋白质,经裂解液(7 mol.L-1尿素,2 mol.L-1硫脲,4%CHAPS,65 mmol.L-1DTT,2%IPG buffer)裂解之后,上样700μg聚焦70 000 Vh,经过SDS-PAGE电泳(分离胶浓度15%,恒功率5 W,电泳时间8 h)后有良好的分离效果,能满足开展后续蛋白质组学研究的实验要求。     

毛竹笋蛋白的双向电泳分析

以毛竹笋为材料,采用改良TCA-酚法提取竹笋总蛋白,Bradford法测定其浓度为9.470 mg·m L-1,采用1 500μg的上样量,使用24 cm,p H 3~10 NL IPG预制胶条进行总蛋白的双向电泳,凝胶图谱经PDquest 8.0软件分析,结果得出,毛竹笋的蛋白点数有1 014个,等电点主要集中在p H 6.0~7.0范围,相对分子质量集中在在45~116 k Da范围内,占58.48%。     

4种黄皮叶片蛋白质提取方法的比较

为了研究不同提取方法对黄皮叶片蛋白质提取质量的影响,以‘杨妃山黄皮’的叶片为材料,采用TrisHCl法、三氯乙酸(TCA)/丙酮法、尿素(Thi)/硫脲(Urea)法、酚(Phe)-甲醇/醋酸铵沉淀法提取叶片组织蛋白质,对获得的蛋白质产量、单向电泳图谱和双向电泳图谱分析比较。结果显示,不同方法的提取效果各异,就提取的蛋白质产率、单向电泳以及双向电泳结果来说,三氯乙酸(TCA)/丙酮法提取效果较理想,单双向电泳图谱辨识度强,蛋白点数多、蛋白点整洁、且纵横纹少。说明该法不但可以较好地去除黄皮叶片中存在的非蛋白物质,而且可以获取高质量的蛋白质。     

氯化钙、ABA和蔗糖预处理对解除休眠水曲柳种子低温脱水萌发的影响

【目的】比较氯化钙、ABA和蔗糖预处理对解除休眠水曲柳种子低温脱水萌发的影响,并探索在不同预处理条件下脱水过程中种子内部的生理变化,为揭示种子脱水耐性调控的生理机制提供参考依据。【方法】以解除休眠的水曲柳种子为材料,利用不同浓度(10~(-2),10~(-3),10~(-4),10~(-5) mol·L~(-1))的氯化钙溶液、不同浓度(10-3,10-4,10-5,10-6mol·L~(-1))的ABA溶液或不同质量浓度(1,10,50,100 g·L~(-1))的蔗糖溶液浸泡种子24 h,以蒸馏水浸泡种子24 h为对照,比较不同处理种子在低温(5℃)条件下干燥脱水后的萌发能力,确定几种预处理措施对种子低温脱水萌发的影响。再依据上述试验结果,分别选择出提高种子萌发能力最有效的处理浓度:氯化钙10-3mol·L~(-1)、ABA 10-6mol·L~(-1)、蔗糖100 g·L~(-1),以经上述溶液预处理24 h后低温脱水的种子为材料,以蒸馏水浸泡24 h后低温脱水种子为对照,进行种子浸出液相对电导率、种子抗氧化酶(SOD、POD和CAT)活性、丙二醛(MDA)含量、可溶性糖、可溶性蛋白和淀粉含量测定,分析不同预处理措施对种子细胞膜透性、抗氧化系统酶活性和贮藏物质代谢的影响。【结果】10-3mol·L~(-1)氯化钙或10-6mol·L~(-1)ABA预处理可显著提高种子在低温(5℃)下脱水后的发芽率和发芽指数,缩短发芽时间;100 g·L~(-1)的蔗糖预处理可显著提高种子在低温(5℃)下脱水后的发芽率,但在提高发芽指数和缩短发芽时间上并没有明显效果。10-3mol·L~(-1)氯化钙预处理的种子,胚中SOD活性在低温脱水后显著提高,种子相对电导率和MDA含量显著降低;10-6mol·L~(-1)ABA预处理的种子,低温脱水后胚中POD活性明显提高,种子相对电导率和MDA含量显著降低;100 g·L~(-1)蔗糖预处理的种子,低温脱水后胚中SOD和POD活性均显著提高,种子相对电导率和MDA含量显著降低。氯化钙、ABA或蔗糖预处理,显著降低了种子胚和胚乳中可溶性蛋白的含量,显著提高了胚中可溶性糖的含量。【结论】适宜浓度的氯化钙(10-3mol·L~(-1))、ABA(10-6mol·L~(-1))和蔗糖(100 g·L~(-1))预处理(尤其是氯化钙或ABA预处理)能够提高水曲柳种子低温脱水后的萌发能力。氯化钙、ABA和蔗糖预处理,会显著提高低温脱水条件下种子胚中的抗氧化系统酶活性,更好地维持细胞膜结构的完整性,降低细胞的离子外渗和MDA含量。但不同预处理种子胚中的抗氧化酶活性表现不同,氯化钙预处理提高胚中SOD活性的作用更明显,ABA预处理提高胚中POD活性的作用更明显,而蔗糖预处理则同时提高胚中的SOD和POD活性。氯化钙、ABA和蔗糖预处理后低温脱水过程中种子胚中可溶性糖含量显著增加,这可能是种子低温条件下脱水保护功能提高的原因之一。     

白蜡及白蜡高级烷醇对人真皮乳头细胞生长的影响

[目的]明确白蜡及白蜡高级烷醇吐温水溶液对人真皮乳头细胞(HFDPCs)生长的影响。[方法]采用台盼蓝法测定细胞的的接种数量,接种第2天给药,给药48 h后采用MTS法研究不同浓度吐温80、白蜡及白蜡高级烷醇吐温水溶液对人真皮乳头细胞的作用。[结果]人真皮乳头细胞在吐温80用量1.56 6.25μg·m L~(-1)范围内能正常生长。在安全范围内,白蜡(增溶量为0.31 1.25μg·m L~(-1))吐温水溶液、白蜡高级烷醇(增溶量为3.12 12.5μg·m L~(-1))吐温水溶液均能促进人真皮乳头细胞的增殖,其中,水溶液中含有6.25μg·m L~(-1)吐温80和1.25μg·m L~(-1)白蜡、水溶液中含有6.25μg·m L~(-1)吐温80和12.5μg·m L~(-1)白蜡高级烷醇时细胞的增殖率明显高于非那雄胺。[结论]吐温80作为增溶剂,其用量低于6.25μg·m L~(-1)时对人真皮乳头细胞安全。在保证吐温80对细胞安全的条件下,白蜡及白蜡高级烷醇可显著促进人真皮乳头细胞的增殖。     

勃氏甜龙竹笋蛋白的提取及双向电泳分析

为建立一套适于勃氏甜龙竹笋蛋白的双向电泳体系,分别采用TCA/丙酮法和改良TCA-酚法提取竹笋总蛋白,pH 3～10和pH 4～7的IPG胶条进行蛋白双向电泳研究。结果表明,TCA/丙酮法更适合勃氏甜龙笋总蛋白的提取,不仅具有更高的提取率,而且还能提取更多的蛋白种类;采用24 cm,pH 4～7线性IPG胶条,共分离出约1 196个蛋白点,比pH 3～10胶条获得更多的蛋白点数量,且蛋白点清晰可见,分辨率更高,分离效果更好。勃氏甜龙竹的竹笋蛋白主要是以等电点在pH 4～7,分子量集中在10～40 kDa范围内的低分子量酸性蛋白为主。     

外源ABA对短期H_2O_2胁迫下侧柏幼苗活性氧代谢及相关基因的影响

[目的]研究外源ABA处理对H_2O_2胁迫下侧柏幼苗活性氧代谢系统的影响,探讨ABA调控侧柏氧化胁迫的可能作用机制。[方法]以侧柏幼苗为试验材料,采用水培方式,研究外施低浓度(0.5μmol·L~(-1))和高浓度(200μmol·L~(-1))ABA对100 mmol·L~(-1)H_2O_2胁迫下侧柏幼苗活性氧代谢的影响。[结果](1)100 mmol·L~(-1)H_2O_2胁迫48 h显著增加了侧柏幼苗叶片过氧化氢(H_2O_2)、丙二醛(MDA)、谷胱甘肽(GSH)和脯氨酸含量、抗氧化物酶(SOD和CAT)活性,而可溶性蛋白含量降低。(2)相较于高浓度200μmol·L~(-1)ABA,施加0.5μmol·L~(-1)ABA显著减少了H_2O_2胁迫下侧柏幼苗H_2O_2和MDA的积累,进一步提高了侧柏幼苗叶片SOD、POD和CAT活性,同时促进GSH、脯氨酸和可溶性蛋白的合成。(3)100 mmol·L~(-1)H_2O_2胁迫处理48 h,侧柏幼苗叶片活性氧代谢相关基因Cu/Zn-SOD、CAT、GR、APX、MDAR和GST表达水平较对照CK均有显著性提高;正常和H_2O_2胁迫下侧柏幼苗外施0.5μmol·L~(-1)ABA相较于200μmol·L~(-1)更有利于提高侧柏叶片活性氧代谢相关基因Cu/Zn-SOD、CAT、GR、APX、MDAR和GST的表达量。[结论]低浓度0.5μmol·L~(-1)ABA有效地增强抗氧化系统的防御能力,减弱幼苗的氧化胁迫和膜脂过氧化水平,从而降低活性氧对侧柏的伤害。     

银杏花粉离体萌发培养基的优化

为优化银杏花粉萌发培养基,采用离体萌发法,通过单因素试验和正交试验分析培养基中不同营养成分对银杏花粉萌发的影响。结果表明:在单因素试验中,蔗糖、硼酸和氯化钙在适宜质量浓度范围内促进花粉萌发,过高质量浓度反而不同程度地抑制花粉萌发;在正交试验中,蔗糖、硼酸和氯化钙对银杏花粉在培养基上离体萌发的影响表现极显著,适宜质量浓度分别为100 g·L-1、10 mg·L~(-1)、40 mg·L~(-1)。     

麻竹叶片蛋白质双向电泳体系的建立

以麻竹花药培养再生植株叶片为材料,比较了改良酚抽法、三氯乙酸(TCA)-丙酮沉淀法和裂解液法3种不同方法对麻竹蛋白质双向电泳的影响,并对上样量、等电聚焦程序、平衡时间等进行了探索与优化.结果表明,用改良酚抽法分离麻竹叶片蛋白质、确定上样量为1 mg· IPG胶条-1、等电聚焦总聚焦功率为100 000 W、胶条两步平衡时间均设置为15 min、考马斯亮进行凝胶染色的实验体系可以获得分辨率高、重复性好的双向电泳图谱,凝胶的蛋白斑点匹配率平均为85.57％,总蛋白斑点数的相对标准差为4.86％.本研究建立了一套适合麻竹叶片蛋白质组学分析的双向电泳实验体系,为进一步研究麻竹生长发育分子调控机制及其遗传改良奠定基础.     

抗松材线虫病赤松体细胞胚的发育和成熟萌发

【目的】研究脱落酸、PEG、肌醇、碳源、凝固剂及培养方式等对抗松材线虫病赤松体细胞胚发育及成熟萌发的影响。【方法】通过对抗松材线虫病赤松种质资源库的调查与分析,选用诱导和增殖状态良好的抗松材线虫病赤松无性系22#-1和13#-1为供试材料,通过不同培养条件的对比试验,筛选出最优的抗病赤松体细胞胚发育及成熟萌发条件。采用SPSS17.0等软件对体细胞胚进行统计分析。【结果】添加15 mg·L~(-1)ABA和140 g·L~(-1)PEG8000的LP培养基上,正常体细胞胚数达到171个·g~(-1),高于其他处理;在渗透剂方面,肌醇的浓度为8.0g·L~(-1)时,抗病赤松的正常体细胞胚数最多,达到179个·g~(-1),胚性愈伤组织直接放置于添加了肌醇的成熟培养基中,没有在中间过渡培养基中培养,因此可以缩短2~3周培养时间,在8周前后就形成了成熟的子叶胚;在碳源方面,添加60 g·L~(-1)麦芽糖的LP培养基上,正常的体细胞胚数达到235个·g~(-1),高于其他处理;在凝固剂方面,植物凝胶浓度为3.0 g·L~(-1)时,培养基凝固,软硬适中,产生的子叶胚生长良好,而培养基中添加了聚乙二醇(PEG),凝固剂琼脂从6.0 g·L~(-1)添加至12.0 g·L~(-1)时,培养基均不能凝固,愈伤组织无法生长,也无子叶胚形成;在培养方式方面,采用液-固增殖-固成熟方式培养时,胚性愈伤组织经11~12周发育为成熟体细胞胚,其中22#-1的正常体细胞胚数最多达到239个·g~(-1),高于其他处理;在体细胞胚萌发培养基上产生的体细胞胚的萌发率和植株转化率分别为67.2%和46.5%,再生植株移栽3个月后成活率为32.7%。【结论】将抗松材线虫病赤松胚性愈伤组织置于15.0 mg·L~(-1)ABA、140 g·L~(-1)PEG8000、8.0 g·L~(-1)肌醇、60 g·L~(-1)麦芽糖、3.0 g·L~(-1)植物凝胶的LP培养基上,以液-固增殖-固成熟培养方式培养,成功获得了正常的体细胞胚和再生植株,同时可缩短2~3周培养时间,加快抗松材线虫病赤松体细胞胚胎发生的进程。研究结果可为抗病赤松的大规模繁殖和工厂化生产提供技术支持。     

Improving the evaluation of multiple-dowel-type connection strength

Our study focuses on predicting the ultimate short-term load carrying capacity of timber-to-timber connections with dowel-type fasteners. The wide range of possible configurations in practice makes the resolution of these values by tests unrealistic. Moreover, different current regulations do not consider some specific failure mechanisms. In many countries, the reduction of resistance involved by this phenomenon is taken into account by considering an effective number of dowels (n_ef) smaller than the actual number of dowels (n) in the connection. However, these different regulations disagree on the values of n_ef and on other points (spacing, partial coefficient of security, formulas). These discrepancies in design rules invite the fundamental research on this topic and, therefore, new methods are sought in order to estimate the load carrying capacities of the connections. In the light of these, our approach consists of predicting the load carrying capacity with the neural network numerical tool. The results obtained by this analysis tool are satisfactory, although the model remains complex. Subsequently, we focus on the simplification of this numerical model with classical regression techniques in order to implement it in a design code.     

<Emphasis Type="Italic">Ophiostoma</Emphasis> species associated with bark beetles infesting three <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Ophiostoma species were isolated from bark beetles and Abies mariesii, A. veitchii and A. homolepis attacked by the beetles in Nikko, Tochigi, central Honshu, Japan. One to two Ophiostoma species were frequently isolated from each species of bark beetle. Ophiostoma subalpinum was the most common associate of Cryphalus montanus. Ophiostoma sp. B as well as O. subalpinum was a common fungus associated with Polygraphus proximus. Ophiostoma europhioides was isolated from Dryocoetes hectographus and D. autographus as one of the common associates. Ophiostoma sp. J and Ophiostoma sp. S were frequently isolated from D. autographus and D. striatus, respectively. These fungi seem to have specific relationships with particular bark beetles. Ophiostoma sp. B, Ophiostoma sp. J and Ophiostoma sp. S have unique morphological characteristics and appear to be new species. Five trees of A. veitchii, approximately 43 years old, were inoculated with five Ophiostoma species to assess the relative virulence of the fungi. Ophiostoma subalpinum, Ophiostoma sp. B, and O. europhioides had relatively higher virulence than the other species studied.     

Parasitic Ichneumonoidea on the horse chestnut leaf miner, Cameraria ohridella (Lepidoptera: Gracillariidae) in Slovakia

Parasitic Hymenoptera from the suprafamily Ichneumonoidea attacking the horse chestnut leafminer Cameraria ohridella Deschka & Dimic (Lepidoptera: Gracillariidae) were studied in southwest Slovakia during 2000 and 2001. The search for such species resulted in the determination of eight ichneumonid species, Gelis agilis (Fabricius), Itoplectis alternans (Gravenhorst), I. clavicornis (Thompson), I. maculator (Fabricius), Scambus annulatus (Kiss), S. brevicornis (Gravenhorst), Zatypota percontatoria (Müller), Adelognathus sp. and a braconid Colastes braconius Haliday. The most abundant species was S. annulatus. All these species recorded are polyphagous and often facultative hyperparasitoids, except C. braconius, which departs from this norm in not being hyperparasitic. The presence of more diversified native parasitoids and hyperparasitoids from the Ichneumonoidea predicate a slow successive adaptation of the parasitoid fauna to the invasive host, Cameraria ohridella.     

Decomposition and nutrient loss from prunings of different contour hedgerow species in tea plantations in the sloping highlands of Sri Lanka

Contour hedgerows of multipurpose tree species in the sloping tea lands of Sri Lanka are expected to reduce soil erosion and also add significant amounts of plant nutrients to the soil via periodic prunings. The objective of this experiment was to characterize the biomass decomposition pattern and quantify the amount of nutrients added through prunings of six tree species (Calliandra calothyrsus, Senna spectabilis, Euphatorium innulifolium, Flemingia congesta, Gliricidia sepium and Tithonia diversifolia) currently being used in hedgerows associated with tea. Withered leaf and stem prunings (50 g) were enclosed in 2-mm litter bags, placed at 5-cm depth and retrieved after one, three, six, nine and 12 weeks. Loss of initial dry weight, N, P and K was measured. Single exponential decay function adequately described both dry weight and nutrient loss. Tree species differed significantly in their rate of breakdown with decomposition constants (k) varying from 0.0299 to 0.2006 week⁻¹ for leaves and from 0.0225 to 0.0633 week⁻¹ for stems. Gliricidia showed the highest k for leaves with the rest in the following descending order: Senna > Tithonia ≥ Euphatorium > Calliandra > Flemingia. A similar pattern was observed for loss of all nutrients with Calliandra and Flemingia always having lower k values than the rest. Although N immobilization was not observed, immobilization of P and K was observed during the first week of incubation in some species, particularly in stem prunings. Annual biomass of prunings differed significantly between tree species in the following descending order: Calliandra > Senna > Flemingia > Tithonia > Gliricidia > Euphatorium. Calliandra added the greatest amount of nutrients annually to the soil with Euphatorium adding the least. Calliandra prunings provided the annual total K requirement and 49% of the N requirement of mature tea. However, none of the species provided more than 5% of the P requirement. It is concluded that among the tree species tested, Calliandra and Flemingia are the most suitable for contour hedgerows in tea plantations of this agroclimatic region because of their higher soil nutrient enrichment capacity and slower decomposition rates which would minimize leaching losses. This revised version was published online in June 2006 with corrections to the Cover Date.     

Potential to harness superior nutritional qualities of exotic baobabs if local adaptation can be conferred through grafting

Baobab leaves form an important part of the local diet in Sahel countries and elsewhere in Africa. Existing leaf nutritional data and agroforestry performance information are based solely on Adansonia digitata L., the baobab of continental Africa. The introduction potential of Adansonia species from the center of diversity in Madagascar and from Australia remains untapped. To assess this potential, the mineral contents and B₁ and B₂ vitamin levels of dried baobab leaves were determined for five-year old trees of A. digitata, A. gibbosa (A. Cunn.) Guymer ex D. Baum, A. rubrostipa Jum. & H. Perrier (syn. A. fony Baill.), A. perrieri Capuron and A. za Baill. grown in an introduction trial in Mali. Nutritional data were evaluated against survival and vigor to identify promising germplasm. Leaf vitamin and crude protein contents were highest in the Madagascar species, especially A. rubrostipa (B₁ 88 mg 100 g⁻¹, B₂ 187 mg 100 g⁻¹, protein 20.7% dry weight). However, the local species far outperformed the introductions in survival, tree height, basal diameter and resistance to termites. We suggest grafting as a way of harnessing the vigor of well-adapted local baobab varieties to the superior nutritional profiles of A. rubrostipa and others. Cross-species grafting tests in Adansonia were successful, thus creating new agroforestry possibilities with different scion/rootstock combinations.     

Insecticidal activity of certain medicinal plants

The methanol extracts of eight species of medicinal plants were tested for insecticidal activity in third instar larvae of Egyptian cottonworm (Spodoptera littoralis). All extracts showed a certain degree of larval toxicity. The extracts of Ocimum basilicum, Origanum majorana and Salvia officinalis appeared to be highly toxic. The extracts significantly affected the growth indexes [relative growth rate (RGR), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD)].     

A new norlignan from Taxodium ascendens

A new norlignan, (2R,3R,4S,5S)-2,4-bis(4-hydroxyphenyl)-3,5-dihydroxy-tetrahydropyran (1), together with 9 known compounds were isolated from the branches and leaves of Taxodium ascendens. Their structures were mainly determined on the basis of MS, IR, 1D and 2D NMR spectral evidences. Methanol extract showed inhibitory activity on carbonic anhydrase II with an IC₅₀ value of 4.27 µg/ml, acetone extract and methanol extract inhibited activity of cathepsin B with IC₅₀ values of 2.12 and 3.71 µg/ml, respectively.     

Rehabilitation of diseased cacao fields in Peru through shade regulation and timing of biocontrol measures

The objective of this case study was to test the effectiveness of biocontrol for the rehabilitation of abandoned, diseased (moniliasis, witches' broom and black pod) cocoa and to develop testable hypotheses regarding the effect of shade and biocontrol timing. Three trial fields differed in their shade regimes. Biocontrol was administered starting either at flowering or pod filling. Disease incidence and yield were compared with cultural control alone. Biocontrol reduced moniliasis from 49% to 25%, independent of the shading regime and application time. Witches' broom was most severe without shade and unaffected by biocontrol. No significant effects on black pod were observed. Although biocontrol increased percentage healthy fruit independent of shading and timing, only the shaded plot with biocontrol from flowering onwards responded with increased yield. Our hypothesis is: 'In the rehabilitation of abandoned cacao, only fields under sustainable shade management and early onset of biocontrol can realise their production potential.' This revised version was published online in June 2006 with corrections to the Cover Date.     

Populations of adult fungus gnats and shore flies in British Columbia container nurseries as related to nursery environment,and incidence of fungi on the insects

Adult fungus gnat and shore fly populations were monitored in four container nurseries near Victoria, British Columbia. In general, populations of both insects were larger in summer than in winter. Also, both insects were more abundant in the nursery with a plastic-covered, soil floor, which favored moisture accumulation and build up of algae, liverworts and mosses, than in nurseries with other types of floors such as cement. Results of fungus isolations revealed that gnats and flies both harbor the seedling pathogens Botrytis cinerea and species of Fusarium and Phoma, plus several non-pathogenic fungi. The results are discussed in relation to nursery sanitation practices, their effects on gnat and fly populations, and some possible roles of these insects in seedling disease epidemiology.     

<Emphasis Type="Italic">Bursaphelenchus mucronatus</Emphasis> (Nematoda: Aphelenchoididae) vectored by <Emphasis Type="Italic">Monochamus </Emphasis><Emphasis Type="Italic">urussovi</Emphasis> (Coleoptera: Cerambycidae) in Hokkaido,Japan

Bursaphelenchus mucronatus Mamiya et Enda has been recovered for the first time from adults of the cerambycid beetle, Monochamus urussovi (Fischer), in Hokkaido, Japan. The nematode was also recovered from the inner bark of Picea jezoensis (Siebold et Zuccarini) Carrière and Abies sachalinensis (Fr Schmidt) Masters infested with M. urussovi larvae. PCR–RFLP analysis indicated that B. mucronatus in Hokkaido is the European type.