四倍体刺槐枝条韧皮部总蛋白质双向电泳体系建立及应用

采用TCA/丙酮法对四倍体刺槐枝段韧皮部全蛋白质进行提取,通过对IPG胶条pH梯度、分离胶浓度的选择,上样量、等电聚焦条件的优化,建立起四倍体刺槐枝段韧皮部蛋白质双向电泳体系。研究结果表明:采用TCA/丙酮法提取四倍体刺槐枝段韧皮部全蛋白质,选用pH4-7的17 cm IPG胶条,考马斯亮蓝染色上样量550μg,等电聚焦IEF聚焦总伏小时数从60 000 Vh提高到80 000 Vh,并采用12%的分离胶对四倍体刺槐枝段韧皮部全蛋白进行双向电泳,能得到背景清晰、蛋白质点数相对较多,分离度高且重复性好的电泳图谱。利用建立的体系进行双向电泳分离蛋白质,能直接挖点送质谱分析。采用该体系分析四倍体刺槐硬枝扦插生根愈伤组织阶段蛋白质表达差异,共筛选出83个差异蛋白质点,其中上调蛋白15个,新产生蛋白22个,下调蛋白22个,缺失表达24个。     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率(浓度)高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

柑橘叶片和果皮总蛋白质提取及双向电泳体系的建立

以春甜橘(Citrus reticulata Blanco‘Chuntianju’)果皮和叶片为材料,分别用Tris-HCl、尿素/硫脲(Thi/Urea)、三氯乙酸/丙酮(TCA)和酚(Phe)等4种方法提取柑橘总蛋白质,从蛋白质产量、单向SDS-PAGE和双向电泳等方面进行比较。结果表明,4种方法的分离效果存在较大差异,不论是以柑橘叶片还是果皮为材料,均以TCA法最好,且双向电泳图谱分辨率较好,蛋白点清晰、均匀、基本没有条纹,且蛋白点多。这说明TCA法不仅能很好地去除柑橘果皮、叶片中存在的大量干扰物质,而且还能得到稳定的蛋白点。     

甜瓜叶片中Rubisco的去除及双向电泳体系的优化

为降低Rubisco的干扰,建立适合于甜瓜叶片蛋白质的双向电泳技术,本文比较了不同全蛋白提取方法和上样量对双向电泳的影响。结果表明,Mg/NP-40/PEG3350/TCA/丙酮提取法可去除样品中绝大多数的Rubisco,使低丰度蛋白得以检测,适合后续分析。以该法提取的蛋白,采用600、800、1000和l200μg四种上样量进行双向电泳,上样量为l000μg的样品用pH3～10的IPG胶条,在2-DE胶上分辨出质量好、数量多的蛋白质点（562个）。因此,Mg/NP-40/PEG3350/TCA/丙酮法是适合甜瓜叶片蛋白质提取的方法,l000μg是最适上样量。     

莲种子蛋白质提取方法比较与双向电泳分析

莲(Nelumbo nucifera Gaertn.)不仅是重要的水生蔬菜作物之一,而且是进行基础研究的好材料。本文采用4种蛋白质提取方法(新型TCA/丙酮法、传统TCA/丙酮法、改良的Tris-HCl法、Tris-饱和酚法)并结合双向电泳技术,对莲子蛋白质提取方法进行筛选与优化。双向电泳实验结果显示,所得蛋白质图谱与莲种子蛋白质组成分布特点一致。通过PDQuest软件分析表明,新型TCA/丙酮法适用于莲子叶和胚芽组织的双向电泳蛋白质提取,而传统TCA/丙酮法则适用于莲胚轴组织双向电泳的蛋白质提取。研究结果为进一步利用质谱进行莲子蛋白质组研究奠定了基础。     

甘蓝型油菜温敏核不育系SP2S花蕾总蛋白质双向电泳体系的建立及应用

以甘蓝型油菜温敏核不育系SP2S及其近等基因系SP2F为材料,分别采用TCA/丙酮法和Tris-丙酮-酚法提取花蕾总蛋白,并优化电泳体系,对可育和不育花蕾中的蛋白质表达进行差异分析。结果表明:TCA/丙酮法适合于花蕾总蛋白的提取,采用10%浓度的分离胶,上样量为450μg,用pH 4~7的17 cm线性胶条和改良的考马斯亮蓝染色法得到了背景清晰、蛋白点分布均匀且重复性好的电泳凝胶图谱。利用这一实验体系,筛选出SP2S和SP2F之间大量的差异蛋白点。相比SP2F,仅在SP2S中表达的有13个点,上调2倍以上的点有4个,下调2倍以上的点有9个。     

适于小麦叶片蛋白质组分析的样品提取方法研究

以‘铭贤169'小麦苗期叶片为材料,分别采用传统的TCA/丙酮沉淀法、酚提取-甲醇/醋酸铵沉淀法以及改进的TCA/丙酮沉淀-酚/SDS联合抽提法提取叶片总蛋白,进行双向电泳分离和胶体考染,以建立适用于小麦蛋白质组分析的样品制备方法.结果表明:TCA/丙酮沉淀法较酚提取-甲醇/醋酸铵沉淀法获得的蛋白杂质较少,在二维电泳图谱中的蛋白点较酚抽提-甲醇/醋酸铵沉淀法提取的蛋白点清晰且多.相比于以上2种提取蛋白样品方法,改进的TCA/丙酮沉淀-酚/SDS联合抽提法提取的小麦叶片蛋白杂质少、二维电泳图谱上的点明显增多、分辨率较高.所选小麦的代表性蛋白点能获得成功鉴定.该方法可推广应用于水稻叶片蛋白质组分析的样品提取.     

利用双向电泳技术分离大豆矮秆突变体相关蛋白

矮秆是大豆育种的重要目标性状之一。本实验以大豆野生型东农42和矮秆突变体东泽11为材料,利用近年来发展起来的双向电泳技术,在蛋白质水平对两个材料的差异蛋白质进行筛选,目的是鉴定与矮秆突变体相关的蛋白,为基因克隆提供依据。通过对酚(Phenol)法与TCA／丙酮沉淀法二种提取方法、100μg和200μg两种加样量、考马斯亮蓝染色和银染两种染色方法的比较,发现用丙酮沉淀法提取叶片可溶性总蛋白、加样量为200μg进行电泳,用考马斯亮蓝染色的效果较好,从而建立了大豆叶片总蛋白双向电泳技术优化体系。用该体系对野生型与突变体叶片全蛋白的差异分析,鉴定出９个蛋白差异点,其中６个上调表达,３个下调表达。     

灵芝子实体原基双向电泳和总蛋白质提取方法的建立

比较了Tris-饱和酚法和三氯乙酸(TCA)/丙酮沉淀法对灵芝子实体原基总蛋白质的提取效果。用Image Master 2D Platinum6.0软件分析两种方法所提蛋白质的双向电泳图谱,分别得到565和273个蛋白质点;(TCA)/丙酮沉淀法在碱性端低分子量区域有些蛋白质斑点存在拖尾现象,Tris-饱和酚法能有效去除样品中的盐分,使蛋白质的聚焦效果更好,蛋白点数增加。Tris-饱和酚法可做为灵芝子实体原基总蛋白质的提取方法并为其他药用真菌总蛋白质的提取提供参考,同时为本实验室后续研究灵芝双向性固体发酵雷公藤的蛋白差异表达奠定了基础。     

芒果叶片双向电泳体系的建立及优化

为建立适用于芒果叶片总蛋白的双向电泳(two-dimensional electrophoresis,2-DE)体系,使用3种方法(尿素/硫脲法,酚法和三氯乙酸/丙酮法)提取芒果叶片蛋白,采用2种水化方式(主动水化和被动水化)和4种样品上样量(1 000μg,1 300μg,1 500μg,1 800μg)进行双向电泳,G-250考染后,通过PDQuest软件对双向电泳图谱进行比较分析。结果表明,三氯乙酸/丙酮法提取效果最好,所得单向SDS-PAGE条带数与2-DE蛋白点数均多于其它2种方法;主动水化对高丰度蛋白点聚焦效果较好;1 500μg蛋白上样量电泳图谱分辨率最高。该方法的建立为开展芒果蛋白质组学研究提供了参考。     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Biological control of three floating water weeds,<Emphasis Type="Italic">Eichhornia crassipes,Pistia stratiotes</Emphasis>, and <Emphasis Type="Italic">Salviniamolesta</Emphasis> in the Republic of Congo

Since 1999, four specific weevils (Coleoptera, Curculionidae) were released in the Republic of Congo against three exotic floating water weeds: Neochetina eichhorniae Warner and N. bruchi Hustache against water hyacinth, Neohydronomus affinis Hustache against water lettuce, and Cyrtobagous salviniae Calder and Sands against water fern. Recoveries of exotic weevils were made from all 24 release sites except one, and all four species have established and spread (up to 800 km for water hyacinth weevils). Within a few years of releases, control of water fern and water lettuce was such that fishing and navigation could be resumed, while reductions of water hyacinth populations were only beginning.     

New species and new combinations in Peruvian Orchidaceae

seventeen new species and combinations are proposed in the generaChondrorhyncha, Cischweinfia, Cochlioda, Eloyella, Encyclia, Kefersteinia, Koellensteinia, Macroclinium, Rodriguezia, Solenidiopsis, andStenia. All new species are illustrated. A key is provided for 2-flowered species ofMacroclinium, PeruvianSigmatostalix, and PeruvianStenia. Solenidium (Solenidiopsis) peruvianum Schltr. is lectotypified.     

安徽牯牛降的丝孢菌Ⅰ

本文报导作者采自安徽枯牛降自然保护区的18种丝孢菌,分属于5个属,其中有3个新种:牛皮冻生尾孢(Cercospora paederiicola),山鸡椒假尾饱(Pseudocercospora litseae-cubebae),鸡血藤生假尾孢(P. millettiicola)和2个中国新纪录。文中对新种进行了描述及绘图,新记录种作了简要说明。研究的标本保存在中国科学院微生物研究所真菌标本室(HMAS)。     

Changes of dung beetle communities from rainforests towards agroforestry systems and annual cultures in Sulawesi (Indonesia)

Little is known about how tropical land-use systems contribute to the conservation of functionally important insect groups, including dung beetles. In a study at the margin of Lore Lindu National Park (a biodiversity hotspot in Central Sulawesi, Indonesia) dung-beetle communities were sampled in natural forest, young secondary forest, agroforestry systems (cacao plantations with shade trees) and annual cultures (maize fields), each with four replicates (n = 16 sites). At each site we used 10 pitfall traps, baited with cattle dung, along a 100 m transect for six 3-day periods. The number of trapped specimens and species richness at the natural forest sites was higher than in all land-use systems, which did not significantly differ. Each land-use system contained, on average, 75% of the species richness of the natural forest, thereby indicating their importance for conservation. However, a two-dimensional scaling plot based on NESS indices (m = 6) indicated distinct dung beetle communities for both forest types, while agroforestry systems and annual cultures exhibited a pronounced overlap. Mean body size of dung beetles was not significantly influenced by land-use intensity. Five of the six most abundant dung beetle species were recorded in all habitats, whereas the abundance of five other species was significantly related to habitat type. Mean local abundance and number of occupied sites were closely correlated, further indicating little habitat specialisation. The low dung beetle diversity (total of 18 recorded species) may be due to the absence of larger mammals in Sulawesi during historical times, even though Sulawesi is the largest island of Wallacea. In conclusion, the dung beetle fauna of the lower montane forest zone in Central Sulawesi appears to be relatively robust to man-made habitat changes and the majority of species did not exhibit strong habitat preferences.     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.