黄瓜器官特异蛋白的研究

利用 SDS单向电泳对黄瓜 ( Cucumissativus L .)的根、茎、叶、花萼、花冠、雄蕊、花柱和子房的可溶性蛋白进行了分析和比较。检测到花冠中的 2 3.5 k D和 33.0 k D,雄蕊中的 1 8.8k D、2 8.5 k D、31 .0 k D、37.0 k D和 39.0 k D,花柱中的 4 5 .0 k D及子房中的 32 .5 k D蛋白 ,分别为各自器官中的器官特异蛋白质。对花冠、雄蕊、花柱和子房的可溶性蛋白的 IEF- SDS双向电泳分析也确定了相应于 SDS单向电泳上特异蛋白带的蛋白质斑点。而且相应于 SDS单向电泳上的一条带 ,在 IEF- SDS双向电泳上可能是一个以上的分子量相同而等电点不同的几个蛋白质斑点。各种器官的蛋白质含量以雄蕊为最高、花萼为最低。     

Expression of Actin Gene During Development of Snake Gourd (Luffa cylindrica) and Cucumber (Cucumis sativum)

Results of Northern blot and Dot blot analysis indicated that actin genes exhibit organ-specific expression in snake gourd (Luffa cylindrica L. ) and cucumber (Cucumis sativum L. ). Actin genes showed obvious developmental specificity during'the development of snake gourd seedlings, mRNA levels in stems of 30-day old seedlings were 4 ～ 6 times higher than that of roots and cotyledons of 8-day old seedlings and roots and hypocotyls of 15-day old seedlings, and were even 10～12 times higher than that of stems and leaves of flowering plants. Actin genes also showed organ-specific expression in young fruits (15 days after flowering) of cucumber.     

水稻胚胎发育时期的特异性蛋白质

对不同发育时期的水稻胚蛋白质,进行了单向SDS聚丙烯酰胺凝胶电泳和双向凝胶电泳分析。单向电泳图中存在20kD左右、胚分化完成期以后特有的蛋白质。根据双向电泳图谱,稻胚总蛋白按其在胚胎发育过程中的动态可分为7类,其中组成性的蛋白质约占85％,在不同发育时期基本不变;具有发育时期特异性的蛋白质约占15％。以分化期胚蛋白质的抗体,对各时期的胚蛋白质进行交叉免疫电泳,所得图谱总体上是相似的,但不同时期的图谱之间出现沉淀峰的峰形、位置和数目的差异。交叉免疫电泳图谱的这种变化可能反映了稻胚发育过程中某些分化期蛋白质发生的变化。     

Gradient flattening of sodium dodecyl sulfate (SDS) and isoelectric focusing (IEF) gels

In addition to our previously reported versatile methods for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [1] and isoelectric focusing [IEF]-gel [2], I have achieved molecular weight gradient flattening of the SDS-polyacrylamide gel and pH gradient flattening of the IEF gel at any segment using the same electrophoresis system. Any crowded gel segment where congregated components are not separated well can easily be widened for good separation and any dispersed gel segment where components are too far can easily be narrowed. Therefore, every gel segment can be used effectively and meaningfully because the gradient curve can be ajusted to any distribution of the components. In the crowded area, any small spots of components which could not be detected previously because of nearby heavy staining or strong radioactivity of an abundant component can be sufficiently separated from the nearby spots in a small gel without sacrificing other areas.     

Derangement of Hypothetical Proteins in Fetal Down's Syndrome Brain

The success of the Human Genome Project (HGP) enables prediction of proteins by computer programs from nucleic acid sequences and for which there is no experimental evidence. Clues for function of hypothetical proteins are provided by sequence similarity with proteins of known function in model organisms. The availability of this bulk of new data is of immediate importance to Down's syndrome (DS) research. DS is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and is characterized by somatic anomalies and mental retardation. In addition, overexpression of chromosome 21 genes is directly or indirectly responsible for mental retardation and other phenotypic abnormalities of DS. To allow insight into how trisomy 21 represents the phenotype of DS, we constructed a two-dimensional protein map and investigated expression of 8 hypothetical proteins in fetal DS (n = 7) and control (n = 7) brains (cortex). Two-dimensional electrophoresis (2-DE) with subsequent in-gel digestion of spots and matrix-assisted laser desorption/ionization (MALDI) spectroscopic identification followed by quantification of spots with specific software was applied. Quantitative analysis of hypothetical protein FLJ10849, hypothetical protein FLJ20113, and activator of hsp90 ATPase homologue 1 (AHA1) revealed levels comparable between DS and controls. By contrast, expression levels of hypothetical protein KIAA1185, hypothetical protein 55.2 kDa, hypothetical protein 58.8 kDa, actin-related protein 3beta (ARP3beta), and putative GTP-binding protein PTD004 were significantly decreased (P < 0.05) in fetal DS brain, and domain analysis suggests involvement in cytoskeleton, signaling, and chaperone system abnormalities.     

利用酵母双杂交系统筛选与黄瓜花叶病毒外壳蛋白互作的寄主蛋白

利用酵母双杂交系统,以黄瓜花叶病毒(Cucumber mosaic virus,CMV)的外壳蛋白(coat protein,CP)为诱饵,从番茄叶片c DNA文库中筛选与其互作的蛋白。结果显示,诱饵载体pBT3-SUC-CMV-CP均能在酵母细胞中正确表达,无自激活活性而且对酵母无毒性;通过对酵母双杂交文库的筛选和回转验证,共获得了98个阳性克隆,分别编码67个可能与CMV-CP相互作用的蛋白,分别参与植物防御反应、光合作用、物质转运、信号转导、能量代谢、氨基酸代谢、细胞壁的形态建成、植物的激素代谢等。本研究结果表明,CMV CP可同时调控寄主的多个代谢过程,在CMV的致病过程中有多重功能。     

乌龙岭’龙眼胚胎发育时期特异性蛋白质的变化

应用IEF-SDS-PAGE技术分析龙眼胚胎分化发育过程中蛋白质组分的变化。结果表明,在各发育阶段大多数蛋白质组分的电泳图谱基本一致,但也有变化。其中花后38d存在TE1(27．1kD、p,7．3),TE2(17．5kD、pI8．2)2个特异蛋白,45d存在TE3(11．4kD、pI7．6),TE4(13．2kD、pI9．9)2个特异蛋白,52d存在TE5(22．6kD、pI7．2),TE6(18．6kD、pI8．3),TE,(23．5kD、pI3．6)3个特异蛋白。31d胚胎电泳图谱中的蛋白质点数相对较多,表明此时蛋白质旺盛合成与积累,这与蛋白含量的变化基本一致。龙眼胚胎发育过程中特异蛋白的出现或消失．对胚胎的分化发育具有重要作用。     

Phylogeny of the Drosophila obscura group as inferred from one- and two-dimensional protein electrophoresis

The phylogenetic relationships of 15 species of the obscura group of Drosophila were analysed by use of one- and two-dimensional electrophoresis. Genetic distances based on two-dimensional data are five times smaller than those based on native proteins. From the data, it is proposed that the species radiation of the obscura group happened in two evolutionary bursts, the first one giving rise to at least four palearctic proto-lineages (bifasciata, obscura (including D. subsilvestris), subobscura, and microlabis) and one or two proto-nearctic lineages (affinis, pseudoobscura), and the second, more recent burst giving rise to the current speciation within lineages.     

Phylogeny of the Drosophila obscura group as inferred from one- and two-dimensional protein electrophoresis

The phylogenetic relationships of 15 species of the obscura group of Drosophila were analysed by use of one- and two-dimensional electrophoresis. Genetic distances based on two-dimensional data are five times smaller than those based on native proteins. From the data, it is proposed that the species radiation of the obscura group happened in two evolutionary bursts, the first one giving rise to at least four palearctic proto-lineages ( bifasciata, obscura (including D. subsilvestris ), subobscura , and microlabis ) and one or two proto-nearctic lineages ( affinis, pseudoobscura ), and the second, more recent burst giving rise to the current speciation within lineages.

Zusammenfassung

Phylogenie der Arten der Drosophila obscura-Gruppe abgeleitet von ein- und zweidimensionaler Protein-Elektrophorese
Die phylogenetischen Verwandtschaftsbeziehungen von 15 Arten der obscura -Gruppe der Gattung Drosophila wurden mit Hilfe von ein-und zweidimensionaler Elektrophorese von Proteinen untersucht. Die genetische Distanzen, die aus den Ergebnissen der zweidimensionalen Elektrophoresen ermittelt wurden, waren fünfmal kleiner als solche, die von nativen Proteinen kommen. Aufgrund der Untersuchungsergebnisse wird angenommen, daß die Radiation der Arten der obscura-Gruppe in zwei evolutiven Schüben erfolgt sei; der erste Schub hätte zu zumindest vier palaerktischen ( bifasciata, obscura mit D. subsilvestris, subobscura und microlabis ) und zwei proto-ne arktischen Linien ( affinis, pseudoobscura ) geführt. In einem zweiten Schub wären dann die endgültigen rezenten Arten entstanden.     

Ciliary Membrane Proteins of Tetrahymena thermophila

SYNOPSIS SDS polyacrylamide gels of the ciliary membrane proteins of Tetrahymena thermophila revealed 5 major peaks and 11 minor protein peaks ranging in molecular weight from below 20,000 to above 250,000. The peaks resembled those found for ciliary membrane proteins of Paramecium aurelia. .     

苜蓿雄性不育植株与可育植株现蕾初期花蕾蛋白质组比较

采用双向凝胶电泳对现蕾初期苜蓿雄性不育植株(Ms-4)及其可育植株(MF)花蕾蛋白质进行了分离,获得了分辨率和重复性较好的双向电泳图谱。通过ImageMaster 2D软件对Ms-4和MF银染图谱分析发现,两者在等电点5~7、分子量20~60 kD范围内蛋白质斑点分布最多,可识别的总蛋白质点数均在6 000个左右,其中差异表达的蛋白质点数为98个;进一步通过质谱分析成功鉴定了22个差异蛋白点。利用Blast2GO程序对 22个蛋白点进行功能注释和代谢途径分析发现,核酮糖羧化酶小亚基、尿苷三磷酸-葡萄糖-1-磷酸尿苷酰基转移酶等蛋白在光合作用、碳水化合物代谢、多细胞生物有机体的发育等过程中起着重要的作用,同时参与了细胞质、细胞壁等组成,并具有绑定、催化、结合和水解等功能。研究结果初步推断,在苜蓿花药发育过程中,蛋白的缺失及表达量的变化可能会使与花粉发育有关的能量缺失,物质合成发生改变,导致雄性不育。     

糠秕马拉色菌酵母态和菌丝态蛋白差异表达的研究

目的通过双向电泳及串联质谱技术鉴定糠秕马拉色菌酵母态及菌丝态差异蛋白,在蛋白水平探讨两态转化机制及致病机理。方法分别诱导糠秕马拉色菌标准株酵母态和菌丝态菌体,利用玻璃珠研磨和超声波破碎细胞壁,三氯乙酸/丙酮沉淀获取总蛋白。双向电泳分离蛋白,PDQuest软件比对找出差异蛋白点。电喷雾串联质谱对差异点进行肽段测序,用Mascot和NCBI的Blast软件经蛋白质数据库鉴定蛋白质。结果经双向电泳分离的糠秕马拉色菌酵母态、菌丝态蛋白各有800多个蛋白点、64个蛋白点表达量有3倍以上差异,其中11个为酵母态特有,9个菌丝态特有。在选取的40个差异点中,成功鉴定出22个点,共16个蛋白。经Mascot和Blast软件检索,有明确功能的蛋白中,肌动蛋白、丝切蛋白等9个蛋白在菌丝态上调,谷胱甘肽转移酶、细胞支架信号蛋白等5个蛋白下调。结论鉴定出16个蛋白分别与细胞代谢、运动、氧化应激等功能相关,为了解糠秕马拉色菌表型转换机制和致病机理提供重要信息。     

Purification of NADPH-specific indole-3-acetaldehyde reductase from Cucumis sativus by two-dimensional native polyacrylamide gel electrophoresis

NADPH-specific indole-3-acetaldehyde (IAAId) reductase from cucumber ( Cucumis sativus L. 相似文献   

黄瓜悬浮细胞蛋白质组双向电泳分析技术

为建立适于黄瓜悬浮细胞蛋白质组分析的双向电泳体系,对黄瓜悬浮细胞蛋白质双向电泳分析所采用的胶条pH范围、样品制备方法、裂解液配方及分离胶浓度等参数进行研究。结果表明,采用pH范围为4～7的IPG胶条,直接裂解后丙酮沉淀法制备黄瓜悬浮细胞蛋白质,裂解液为8mol/L尿素、2mol/L硫脲、2%IPG Buffer、4%CHAPS、1%TBP、65mmol/L DTT、2mmol/L EDTA、0.001%溴酚蓝和1%鸡尾酒,分离胶浓度为11%,可获得蛋白质点分离清晰的双向电泳图谱。     

三种花生幼胚蛋白质提取方法比较研究

植物组织(或细胞)的蛋白质提取效率与效果直接影响蛋白质双向凝胶电泳等实验的结果。为探索建立适用于花生幼胚蛋白质(双向凝胶电泳用)提取的最佳条件,尝试了磷酸缓冲液直接提取法、改良的荔枝胚胎蛋白提取法和Trizol(附加)提取法等3种提取方法,根据蛋白提取得率、试剂成本、双向电泳图谱的质量(蛋白质斑点的丰度、分布特点)进行初步评价。结果表明,磷酸缓冲液直接提取法简单但总体效果较差,改良的荔枝胚胎蛋白提取法综合评价最好,与双向凝胶电泳条件更兼容。     

黄瓜叶片胞间隙蛋白质双向电泳体系的建立

以黄瓜种质‘PI088’幼苗为材料,提取胞间隙液,制备蛋白样品,通过对不同IPG胶条、等电聚焦条件、分离胶浓度、上样量等条件的探索,建立适合黄瓜叶片胞间隙蛋白质组的双向电泳体系.结果显示:(1)用pH 3～10的非线性IPG胶条,等电聚焦时间为70 000 Vh,分离胶浓度为10％,上样量为800 μg时,能够得到较好的2-DE图谱.(2)利用所建立的双向电泳体系找到了对照及接种霜霉菌后2d的黄瓜叶片胞间隙差异蛋白,其中的12个上调表达点和10个下调表达点的表达量变化在1.5倍以上.并选取一个差异点成功进行了质谱分析.(3)质谱分析结果显示,所找的差异点为一种酸性的几丁质酶,等电点为4.27.可见,采用所建立的双向电泳体系可获得分辨率高、重复性好的2-DE图谱并能很好地用于质谱分析.     

Chromatin-Dependent Repression of the Arabidopsis Floral Integrator Genes Involves Plant Specific PHD-Containing Proteins

The interplay among histone modifications modulates the expression of master regulatory genes in development. Chromatin effector proteins bind histone modifications and translate the epigenetic status into gene expression patterns that control development. Here, we show that two Arabidopsis thaliana paralogs encoding plant-specific proteins with a plant homeodomain (PHD) motif, SHORT LIFE (SHL) and EARLY BOLTING IN SHORT DAYS (EBS), function in the chromatin-mediated repression of floral initiation and play independent roles in the control of genes regulating flowering. Previous results showed that repression of the floral integrator FLOWERING LOCUS T (FT) requires EBS. We establish that SHL is necessary to negatively regulate the expression of SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), another floral integrator. SHL and EBS recognize di- and trimethylated histone H3 at lysine 4 and bind regulatory regions of SOC1 and FT, respectively. These PHD proteins maintain an inactive chromatin conformation in SOC1 and FT by preventing high levels of H3 acetylation, bind HISTONE DEACETYLASE6, and play a central role in regulating flowering time. SHL and EBS are widely conserved in plants but are absent in other eukaryotes, suggesting that the regulatory module mediated by these proteins could represent a distinct mechanism for gene expression control in plants.     

黄瓜花叶病毒CP基因原核表达及抗血清的制备

把黄瓜花叶病毒 (CMV)西番莲分离物的外壳蛋白 (CP)基因 ,通过BamHI SacI位点定向插入pET 2 2b( )载体 ,转化大肠杆菌BL2 1 (DE3)中 ,经IPTG诱导下 37℃培养 6h ,SDS PAGE电泳示表达蛋白分子质量为 31 8kDa ,表达量占菌体总蛋白的 2 8 9%,表明该蛋白得到了高效表达。用冰冷的氯化钾溶液显色 ,用表达的特异蛋白质条带制备抗原 ,免疫家兔制备出病毒特异抗血清。采用ID ELISA测定抗血清效价为 1 0 -6;抗血清和CMV几个分离物均有特异反应 ,和TMV、菌体蛋白不发生非特异性反应 ;检测病毒灵敏度达 30ng ml,能够从稀释 31 2 5倍的感病植物汁液中检测出病毒     

采用蛋白质组学技术筛选大肠癌转移相关蛋白

采用对同一亲本来源、不同转移潜能细胞株SW480和SW620的蛋白质表达谱进行双向凝胶电泳和质谱技术分析,并在蛋白质和mRNA水平进行验证,成功鉴定了10个大肠癌转移相关蛋白,其中SW620细胞株表达上调的蛋白质有磷酸甘油酸变位酶1,磷脂酰乙醇胺结合蛋白和高迁移率族蛋白B-1,而热休克蛋白27,膜联蛋白Ⅰ,甲硫腺苷磷酸化酶,切丝蛋白1和表皮型脂肪酸结合蛋白在SW620中表达下调.大多数差异蛋白质功能涉及肿瘤细胞生长、运动、粘附、凋亡等过程,研究结果为阐明大肠癌转移机制及寻找预测大肠癌转移的潜在标志物提供了理论依据.