中华卵索线虫的性别分化分子机理研究进展

中华卵索线虫Ovomermis sinensis Chen et al.是一种宝贵的昆虫天敌资源,具有特殊的寄生期营养竞争压力决定其雌雄性别分化机制。本文重点概述了近年中华卵索线虫性别决定与分化、寄生期生理生化以及分子系统学研究等方面的进展,并对中华卵索线虫今后的研究方向提出了建议。     

中华卵索线虫的研究与应用

中华卵索线虫Ovomermis sinensis Chen etal.作为一种昆虫病原线虫,在农业害虫的生物防治中起着重大作用。文章综述90年代以来我国有关中华卵索线虫的生物学特性、田间应用、体内外培养以及生化与分子生物学研究进展,并对中华卵索线虫今后的研究方向提出建议。     

中华卵索线虫雌雄寄生后期幼虫基因差异表达分析

索科线虫是一类具有很大生防潜力的昆虫(如棉铃虫等)天敌资源,但由于其体外培养尚未获得成功,阻碍了商业化生产和大规模应用,究其原因主要是体外培养的线虫不能完成雌雄性别分化。因此,研究该类线虫性别分化机制成为近年本领域的热点课题。该文以中华卵索线虫为实验材料,采用mRNA差异显示的方法,分析了中华卵索线虫性别分化关键时期雌、雄寄生后期幼线虫的基因表达差异。在雌雄线虫体内得到具有表达差异的基因片段20条。其中8条在雄虫体内特异性表达,12条在雌虫体内特异性表达。利用信息生物学技术对所分离到的差异片段进行了序列分析。其中,Ensembl分析发现4个片段与秀丽线虫X染色体具有可匹配片段,推测这些片段可能是影响中华卵索线虫性别分化的重要基因。这些结果将为后续研究提供思路。     

中华卵索线虫感染棉铃虫后脂肪体结构的变化

采用组织超薄切片技术,电镜下观察研究昆虫棉铃虫Helicoverpa armigera Hǖbner脂肪体细胞的超微结构,结果表明。在中华卵索线虫Ovomermis sinensisChen感染棉铃虫后的不同寄生时期内,宿主脂肪体细胞内的线粒体、内质网、脂肪球和细胞核等细胞器在形态和结构上均有较大的病理变化,这些病理变化,与这一时期中华卵索线虫的生长发育和寄生生活等特点密切相关。     

昆虫病原线虫研究概况

昆虫病原线虫 (ｅｎｔｏｍｏｐａｔｈｏｇｅｎｉｃｎｅｍａｔｏｄｅｓ)是本世纪发展起来的一种有潜能的生物防治因子[1] 。它具有寄主范围广、能主动寻找寄主、对人畜及环境安全无毒 ,并能人工大量培养等优点。因此 ,在农药污染日益严重、害虫抗药性发展迅速的今天 ,昆虫病原线虫已成为可持续发展农业的迫切需要 ,受到国际生防领域的重视。特别是斯氏科线虫和小杆科线虫 ,已成为当前国际生防领域研究热点之一。本文简要概述了线虫的生物学特性、分类及致病机理等方面的研究 ,并结合目前存在的问题探讨了昆虫病原线虫的研究方向和发展前景。1…     

寄生于粘虫的卵索线虫属一新种——中华卵索线虫(线虫纲:索科)

本文报道从河南省驻马店地区和山东省临沂地区麦田内采集的粘虫幼虫的寄生线虫优势种卵索线虫属一新种,命名为中华卵索线虫,新种Ovomermis sinensis sp.nov.     

五种索科线虫RAPD亲缘关系分析

采用RAPD技术构建了索科线虫4属5种的指纹图谱。从47个引物中筛选出12个稳定性好、多态性高的引物,共扩增出161条谱带,其中150条谱带具遗传多态性,占93·17%。所获片段长度大小为200~3200bp,单个引物扩增的条带数在11~16之间,平均为13·42条。采用RAPDistance软件及MEGA程序,计算Nei氏相似系数和遗传距离,建立UPGMA和NJ聚类图,两个聚类图拓扑结构相同,将5种索科线虫分为两大分支:同属于蚊幼寄生罗索属线虫的食蚊罗索线虫(Romanomermisculicivorax)与武昌罗索线虫(R.wuchangensis)亲缘关系最近,先聚在一起,再与同翅目(Homoptera)寄生长沙多索线虫(Agamermischang-shaensis)聚为一支;鳞翅目(Lepidoptera)寄生中华卵索线虫(Ovomermissinensis)和同翅目寄生两索属线虫(Amphimermissp·)亲缘关系较近,两者聚为一支。5种索线虫属内种间的遗传距离较小,食蚊罗索线虫与武昌罗索线虫之间遗传距离仅为0·1789;而属间遗传距离较大,在0·4471~0·5488之间。上述结果表明:RAPD技术可以应用于索科线虫亲缘关系的分析,能够反映出不同线虫间的遗传差距,从而成功地进行属、种的分类及进化问题研究。     

昆虫群落中天敌间的致死干扰竞争作用

致死干扰竞争作用(lethal interference competition)是近些年来才被人们认识到的更为复杂的种间竞争关系, 是昆虫天敌间竞争的一种极端形式, 广泛存在于寄生性天敌昆虫之间。本文从其定义、 作用机制、 方式及其与害虫生物防治的相互关系几个方面介绍了天敌群落中的这一典型的种间相互关系。根据作用机制的不同, 可将致死干扰竞争作用分为外竞争和内竞争; 其通常的作用方式包括多寄生(超寄生)、 复寄生、 杀卵作用、 寄主取食、 物理攻击和生理抑制等。深入全面地研究这一种间关系对于有效生防作用物的筛选和引入, 以及整个生防系统群落的稳定性具有重要意义。     

两种赤眼蜂对小菜蛾卵的寄生潜能分析

应用生命表技术分析了拟澳洲赤眼蜂Trichogramma confusum Viggiani(T.c)和卷蛾分索赤眼蜂Trichogramm-atoidea bactrae Nagaraja(T.b)在两种繁蜂条件组配下（分别用米蛾Corcyra cephalomica(Stainton)(RM)卵和小菜蛾Plutella xylostella(L.)(DBM)卵繁育）对小菜蛾卵的寄生潜能分析,结果表明：（1）在相同寄主繁蜂条件下,卷蛾分索赤眼蜂在小菜蛾卵上显示出较强的寄生潜能,米蛾卵上所繁的卷蛾分索赤眼蜂（T.b-RM)和小菜蛾卵上所繁的卷蛾分索赤眼蜂（T.b-DBM)的内禀增长率为0.3509和0.3450,而米蛾卵上所繁的澳洲赤眼蜂（T.c-RM)和小菜蛾卵上所繁的拟澳洲赤眼蜂（T.c-DBM)的仅为0.2391和0.1902,T.b-RM和T.b-DBM的每雌平均寄生卵数为70.75和46.13粒,而T.c-RM和T.c-DBM的仅为64.90和31.73粒,但拟澳洲赤眼蜂的雌蜂寿命较卷蛾分索赤眼蜂更长。（2）在不同寄主繁蜂条件下,同种赤眼蜂对小菜蛾卵的寄生潜能以米蛾卵所繁的仔蜂的各项寄生特性参数（内禀增长率,每雌寄生卵量,净生殖力,平均世代历期和雌蜂寿命）均优于用小菜蛾卵所繁之蜂,米蛾卵是其适宜的中间寄主。（3）长期用中间寄主繁蜂,赤眼蜂对目标寄主表现出一定的不适应性,中间寄主的驯化对赤眼蜂的寄生潜能有不容忽视的削弱作用。     

昆虫病原线虫(斯氏属与异小杆属)应用概况

<正> 在自然界,线虫与昆虫的联系是普遍的。据目前所知,至少有27个科的线虫与16个目近3000种昆虫有联系(Nickole 1984,Poinar 1983),其分布是由寄主昆虫的栖境所决定的。这些线虫对害虫的种群起着自然调节的作用。线虫与昆虫联系的方式各异,使人最感兴趣的是那些能作为害虫生物防治的病原线虫。自从Oldhan(1933)首次提出利用线虫防治害虫以来,应用病原线虫进行害虫生物防治的工作已     

双向电泳比较两种方法获得的水稻叶片蛋白

水稻是研究单子叶植物遗传和分子生物学的模式植物,是研究植物基因组学和蛋白质组学的一个重要的生物材料。双向电泳作为蛋白质组学中蛋白分离的主要方法,其关键步骤在于蛋白质的提取。为了比较不同蛋白提取方法,采用TCA/丙酮沉淀法和Mg/NP-40提取法分别提取水稻叶片总蛋白,用双向凝胶电泳分离两种方法获得的蛋白。结果表明,在双向图谱中,Mg/NP-40提取法分离的蛋白点数较多,RuBisCO大亚基的含量较低,适于分离等电点在5～7范围内、分子量在14～20kD和高于67kD的水稻叶片蛋白质;TCA/丙酮沉淀法分离的蛋白点数较少,适于分离等电点在4～5范围内、分子量在31～67kD内的水稻叶片蛋白质。     

一种经济、简便的双向电泳方法

双向电泳是蛋白质分析的一门较为精确的技术.对电泳方法进行改良并采用普通垂直薄板等电聚焦技术,不仅大大降低了蛋白质双向电泳的成本,而且操作过程简单,是一种用于初步分析蛋白质的较好方法.在中华卵索线虫(Ovomermis sinensis)雌雄成虫可溶性差异蛋白的分析中获得了较满意的结果.这一改良方法的建立,可望促进双向电泳的广泛应用.     

家蚕蛹期雌雄生殖腺蛋白质双向电泳比较分析

分析家蚕Bombyx mori雌雄生殖腺细胞蛋白质,有利于发现性别分化相关的功能蛋白质,探讨生殖腺发育相关基因的表达调控机理。本研究利用蛋白质双向凝胶电泳和图像分析技术,分析家蚕蛹期第2天的雌雄生殖腺细胞蛋白质。结果表明: 在雄蚕生殖腺蛋白质电泳图谱中共检测到435个蛋白斑点,其中特异性蛋白斑点73个,占总蛋白斑点数的16.8％;雌蚕生殖腺的电泳图谱中有417个蛋白斑点,其中特异性蛋白斑点55个,占总蛋白斑点数的13.2％。雌雄能匹配的蛋白斑点有362对,匹配率达85.0％。     

Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinhardtii

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes but structural studies on this part of the proteome are limited. In this study we undertook such an approach to analyse photosynthetic thylakoid membranes isolated from wild-type and mutant strains of Chlamydomonas reinhardtii. Thylakoid membrane proteins were separated by high-resolution two-dimensional gel electrophoresis (2-DE) and analysed by immuno-blotting and mass spectrometry for the presence of membrane-spanning proteins. Our data show that light-harvesting complex proteins (LHCP), that cross the membrane with three transmembrane domains, can be separated using this method. We have identified more than 30 different LHCP spots on our gels. Mass spectrometric analysis of 2-DE separated Lhcb1 indicates that this major LHCII protein can associate with the thylakoid membrane with part of its putative transit sequence. Separation of isolated photosystem I (PSI) complexes by 2-DE revealed the presence of 18 LHCI protein spots. The use of two peptide-specific antibodies directed against LHCI subunits supports the interpretation that some of these spots represent products arising from differential processing and post-translational modifications. In addition our data indicate that the reaction centre subunit of PSI, PsaA, that possesses 11 transmembrane domains, can be separated by 2-DE. Comparison between 2-DE maps from thylakoid membrane proteins isolated from a PSI-deficient (Deltaycf4) and a crd1 mutant, which is conditionally reduced in PSI and LHCI under copper-deficiency, showed the presence of most of the LHCI spots in the former but their absence in the latter. Our data demonstrate that (i) hydrophobic membrane proteins like the LHCPs can be faithfully separated by 2-DE, and (ii) that high-resolution 2-DE facilitates the comparative analysis of membrane protein complexes in wild-type and mutants cells.     

Protein identification of purified particles isolated from spinach thylakoids by deoxycholate electrophoresis

Three thylakoid complexes were isolated by deoxycholate preparative electrophoresis. The protein composition of each fraction was analyzed by SDS analytical electrophoresis. No protein of the PS 1 enriched fraction (fraction 1) was found in the PS 2 enriched fraction (fraction 2) and inversely. The antenna complex (fraction 3) did not have any contamination by proteins of fraction 1 or fraction 2. Fraction 1 was mainly composed of the CP1, the reaction center complex of the PS1, and by low molecular weight proteins, previously found in other PS 1 preparations. Tentative assignments of these proteins are presented; among them are iron sulfur proteins. After analytical SDS electrophoresis of fraction 2, the reaction center complex was dissociated. Nevertheless three proteins of 50 kD, 42 kD and 35 kD were assigned to this complex. Fraction 2 contained also the three cytochromes of the thylakoid membranes: cyt f, cyt b6, cyt b559. Fraction 3 was exclusively composed of one protein pigment complex, CP2.Abbreviations SDS sodium dodecyl sulfate - PS 1 photosystem 1 - PS 2 photosystem 2 - CP1, CP2 protein pigment complexes isolated by SDS electrophoresis - cyt cytochromes - P700 primary electron donor of PS 1 - P680 primary electron donor of PS 2 - DOC deoxycholate - Q primary plastoquinone electron acceptor - CF coupling factor     

Establishment of a near-standard two-dimensional human urine proteomic map

A proteomic map for human urine on two-dimensional (2-D) gels has been developed. Initial studies demonstrated that the urine proteins prepared by conventional methods showed interference and poor reproducibility in 2-D electrophoresis (2-DE). To address this issue, urine samples were dialyzed to remove any interfering molecules. The dialysis of urine proteins and the concentration by lyophilization without fractionation significantly improved the reproducibility and resolution and likely represents the total urine proteins on a 2-D gel. In addition, removing albumin from urine using Affi-Gel Blue helped to identify the low-abundant proteins. Using the developed method, we prepared proteins from urine collected from healthy females and males. The large inter- and intra-subject variation in protein profiles on 2-D gels made it difficult to establish a normal human urine proteomic 2-D map. To resolve this problem, urinary proteins were prepared from the pooled urine collected from 20 healthy females and males, respectively. The established male and female urine proteomes separated on 2-D gels were almost identical except for some potential sex-dependent protein spots. We have annotated 113 different proteins on the 2-D gel by peptide mass fingerprinting (PMF). We propose that the established total urine proteome can be used for 2-DE analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and identification of novel disease-specific biomarkers.     

双向凝胶电泳比较三种常用蛋白质提取方法

组织(或细胞)的蛋白质提取效率直接影响蛋白质双向凝胶电泳(2-DE)的分辨率.为探索建立适用于人乳腺癌细胞株MCF-7蛋白质提取的最佳条件,比较目前在双向凝胶电泳中常用的3种蛋白质提取方法对MCF-7细胞总蛋白的提取效率.MCF-7细胞经培养后,分别采用M-PER试剂、标准裂解液或含硫脲裂解液提取其总蛋白质,然后进行双向凝胶电泳,并根据凝胶上蛋白质斑点的丰度和分布特点判断所得双向电泳图谱的质量,以确定MCF-7细胞蛋白质提取的相对最佳方法.结果显示,M-PER试剂法得到的图谱分辨率较低,蛋白质主要集中分布在分子量15~70kD,pH4.7~6.3的范围内;标准裂解液法得到的图谱分辨率有所提高,蛋白质分布比M-PER试剂法得到的图谱广;硫脲裂解液法得到的图谱是三者中分辨率最高的,尤其是高丰度蛋白和高分子量蛋白分离效果比前两者好.结果表明,在3种常用的蛋白质提取方法中,硫脲裂解液对细胞蛋白质的溶解性最佳,相对更适合于提取MCF-7细胞的蛋白质,并与双向凝胶电泳条件更兼容.     

New approaches for biomarker discovery: the search for liver fibrosis markers in hepatitis C patients

Despite many shortcomings, liver biopsy is regarded as the gold standard for assessing liver fibrosis. A less invasive and equally or more reliable approach would constitute a major advancement in the field. Proteomics can aid discovery of novel serological markers and these proteins can be measured in patient blood. A major challenge of discovering biomarkers in serum is the presence of highly abundant serum proteins, which restricts the levels of total protein loaded onto gels and limits the detection of low abundance features. To overcome this problem, we used two-dimensional gel electrophoresis (2-DE) over a narrow pH 3-5.6 range since this lies outside the range of highly abundant albumin, transferrin and immunoglobulins. In addition, we used in-solution isoelectric focusing followed by SDS-PAGE to find biomarkers in hepatitis C induced liver cirrhosis. Using the pH 3-5.6 range for 2-DE, we achieved improved representation of low abundance features and enhanced separation. We found in-solution isoelectric focusing to be beneficial for analyzing basic, high molecular weight proteins. Using this method, the beta chains of both complement C3 and C4 were found to decrease in serum from hepatitis C patients with cirrhosis, a change not observed previously by 2-DE. We present two proteomics approaches that can aid in the discovery of clinical biomarkers in various diseases and discuss how these approaches have helped to identify 23 novel biomarkers for hepatic fibrosis.