感染大麦黄花叶病毒(BaYMV)的大麦细胞质内含体及细胞器病变的研究

超薄切片电镜观察表明,在感染大麦黄花叶病毒(BaYMV)的大麦(品种“早熟3号”)叶肉细胞中,液泡周围偶而可看到病毒颗粒束,在发病后期黄化或坏死的叶肉细胞中,可见到散布的病毒颗粒。在所有表现症状的病叶叶肉细胞,表皮细胞和木质部薄壁细胞中均可观察到风轮体、束状体、板状集结体以及膜状体等细胞质内含体,未见 卷简体和细胞核内含体。感病初期细胞中,细胞质丰富,核糖体数量增加,内质网肥大,随着病毒症状发喂,叶绿体、线粒体等细胞器逐渐肿大,外膜破裂直至解体。     

大麦和玉米叶片叶绿体中Rubisco及其活化酶的免疫金标记定位

运用免疫金标记电镜技术研究了禾本科C3植物大麦(Hordeum vulgare L.)和C4植物玉米(Zea mays L.)叶片中Rubisoo及其活化酶(RCA)的细胞定位,结果表明:两种植物叶片解剖结构及叶绿体超微结构差别明显.在大麦叶细胞中,只有一种叶肉细胞叶绿体,Rubisoo和RCA主要分布于叶绿体的间质中.在玉米叶细胞中,存在着维管束鞘细胞和叶肉细胞两种类型叶绿体,Rubisco主要分布于鞘细胞叶绿体的基质中,但在叶肉细胞叶绿体中亦有少量特异性标记;RCA在鞘细胞叶绿体和叶肉细胞叶绿体的基质中都有分布.两种植物叶绿体结构及光合作用关键酶定位的不同,体现了C3植物和C4植物在光合器结构与功能上的差异.     

Rubisco和RCA在青菜叶绿体中的分布及病毒侵染对其细胞定位的影响

运用免疫金标电镜术观察了青菜叶细胞中光合作用关键酶Rubisco和Rubisco活化酶(RCA)的细胞化学定位,结果显示Rubisco和RCA免疫金颗粒主要分布于薄壁组织叶绿体的间质中,在基粒片层上很少,表皮的气孔保卫细胞和维管束薄壁细胞叶绿体内也有分布,在细胞质及线粒体等细胞器中无特异性分布。同时比较观察了感染芜菁花叶病毒(TuMV)的青菜叶绿体Rubisco和RCA免疫金标记结果,发现病组织中结构尚完整的叶绿体Rubisco和RCA标记率略有下降,而结构严重破坏的叶绿体中两种酶标记率分别仅为正常叶绿体的58．44％和64．67％,表明病毒侵染可导致Rubisco和RCA含量下降,影响寄主植物的光合作用。     

Rubisco和RCA在青菜叶绿体中的分布及病毒侵染对其细胞定位的影响

运用免疫金标电镜术观察了青菜叶细胞中光合作用关键酶Rubisco和Rubisco活化酶(RCA)的细胞化学定位,结果显示Rubisco和RCA免疫金颗粒主要分布于薄壁组织叶绿体的间质中,在基粒片层上很少,表皮的气孔保卫细胞和维管束薄壁细胞叶绿体内也有分布,在细胞质及线粒体等细胞器中无特异性分布。同时比较观察了感染芜菁花叶病毒(TuMV)的青菜叶绿体Rubisco和RCA免疫金标记结果,发现病组织中结构尚完整的叶绿体Rubisco和RCA标记率略有下降,而结构严重破坏的叶绿体中两种酶标记率分别仅为正常叶绿体的58.44%和64.67%,表明病毒侵染可导致Rubisco和RCA含量下降,影响寄主植物的光合作用。     

用感染病毒细胞超微结构的差异区分RMV及TMV株系

通过透射电镜观察被长叶车前草花叶病毒(RMV)和烟草花叶病毒(TMV)不同株系感染的普通烟叶肉细胞的超微结构,发现两种病毒的粒子分布、内含体类型、被感染细胞超微结构的病变均存在差异.病毒粒子分布有成束、分散、环状、膜包被及角状成层或平行成层排列等类型,存在于细胞质及液泡中,但未见于细胞核、线粒体及叶绿体等细胞器中.内含体的X-小管形状有长杆状、短杆状及颗粒状,数量各异.细胞壁常引起增厚、结构松散及扭曲等变化.叶绿体聚集成堆或分布于细胞边缘,其数量、大小、形状及所含淀粉粒、嗜锇颗粒等存在差异,有些还有颗粒状物质积累.线粒体及内质网等在不同株系间也存在差异.本项研究表明,被感染细胞超微结构的差异可作为RMV和TMV株系区分的依据.     

复合感染建兰花叶病毒和齿兰环斑病毒的兰花超微结构观察及病原物快速鉴定

通过负染和超薄切片观察到蝴蝶兰(Phalaenopsis amabilis)病叶中线状和杆状病毒颗粒。组织切片观察,同时发现两种病毒粒子的典型聚集体:线状粒子的带状聚集体,粒子多层排列,层间呈一定角度或螺旋相叠;杆状粒子的平行、角层状或螺旋型排列聚集体。二种病毒聚集体均出现于薄壁细胞、细胞间隙和输导组织细胞中。感病细胞中叶绿体发育不全;线粒体增生、肿胀甚至空化;细胞核膨大、空化。进一步的多重RT-PCR与病毒核酸序列分析,同时扩增到建兰花叶病毒(CymMV)和齿兰环斑病毒(ORSV)的外壳蛋白基因,与GenBank已知分离物同源性分别达到98%和99%-100%。从细胞和分子层面揭示了蝴蝶兰受CymMV和ORSV复合侵染并可能导致严重病症的事实,分析和明确了感病兰细胞超微结构的病变特征以及田间病症发生的细胞病理学依据。     

植物基因转译产物的定位与加工(续)

植物基因转译产物的定位与加工（续）朱祯（中国科学院遗传研究所）四、进入分泌系统的蛋白在真核细胞中,胞浆蛋白、核蛋白、叶绿体蛋白以及线粒体蛋白主要是在胞浆内游离核糖体上合成的,随后输入到相应的靶细胞器或继续留在胞浆内。除此之外,很大一部分蛋白是在粗糙内质网（Ｅｎｄｏｐｌａｓｍｉｃｒｅｔｉｃｕｌｕｍ,ＥＲ）的核糖体上合成的,并可跨过ＥＲ膜进入到ＥＲ腔（ＥＲｌｕｍｅｎ）内,通过细胞的分泌途径（Ｓｅcretorpathway)最终定位到细胞内的特定部位或分泌到细胞外。     

长叶车前花叶病毒上海分离株外壳蛋白赖氨酸残基的修饰对感染及重组的影响

长叶车前花叶病毒上海分离株外壳蛋白中有二个赖氨酸残基。完整的病毒颗粒或外壳蛋白在pH8.5条件下,只有一个赖氨酸残基与三硝基苯磺酸反应。如果有4M尿素存在,则有两个赖氨酸残基和一个半胱氨酸巯基被修饰。三硝基苯磺酰化的HRV_(sh)病毒颗粒的感染力丧失90%以上。HRV_(sh)的外壳蛋白经三硝基苯磺酸修饰后不能与HRV_(sh)-RNA重建。这些结果清楚地表明两个赖氨酸残基中一个位于病毒颗粒表面,另一个则包在病毒颗粒中。表面那个赖氨酸残基为HRV_(sh)的感染所必需。     

生物细胞中颗粒状多聚磷酸盐细胞器的结构与功能

多聚磷酸盐(polyphosphat,poly P)是一种由数十个或上百个磷酸根聚合而成的生物大分子,以颗粒状、胶体状和溶解状等多种状态存在于各类生物细胞中。生物体中的poly P能够通过分解提供能量;鳌合金属离子来调节细胞内渗透压,维持质膜稳定;与蛋白质或DNA结合稳定其结构,减轻细胞应激损伤。颗粒状多聚磷酸盐细胞器主要指细胞中用于贮存颗粒状poly P、金属阳离子以及蛋白质、氨基酸和少量水等物质的细胞器。在寄生虫细胞中颗粒状聚磷细胞器常称为酸性钙体,而细菌或者其他微生物细胞中则称为异染颗粒,但是随着研究的不断深入,发现酸性钙体和异染颗粒都具有相似的结构特征,遂将其统一定义为颗粒状多聚磷酸盐细胞器。颗粒状聚磷细胞器的发现拓展了生物共同祖先(last universal common ancestor,LUCA)的学说,丰富了原核生物细胞器认知,我们相信该细胞器在生命起源、抗环境胁迫、生物互作和代谢调控等方面具有重要功能,在疾病治疗以及磷生物地球化学循环过程中发挥重要作用。     

应用菜粉蝶颗粒体病毒单克隆抗体对不同株病毒进行抗原分析

应用杂交瘤技术,以大菜粉蝶颗粒体病毒(pbGV)、菜粉蝶颗粒体病毒北京分离株(prGV-801)、武汉分离株(prGVw1-78)和济南分离株(prGV-J)等4株病毒为抗原,获得9株杂交瘤细胞(Fr1～9)。ELISA测定细胞培养上清抗体效价最高达8000,腹水效价最高达450000。9株杂交瘤细胞所分泌的抗体,各呈现株或组特异性,其中pr1～4分别与4个毒株起反应,pr5～8可与2～3个毒株起反应,而Pr9则与所有4个毒株均起反应。据此,认为这4个毒株有抗原性差异。不同毒株间抗原性的差异不仅存在于病毒粒子上,而且也存在于颗粒体蛋白上。讨论了昆虫病毒单克隆抗体在鉴别病毒抗原性差异,生物防治和流行病学研究中的意义。     

Barley mild mosaic virus inside its fungal vector, Polymyxa graminis

In an electron microscope study to investigate the association of barley mild mosaic virus (BaMMV) with its fungal vector, Polymyxa graminis, thin sections were made of zoospores of the vector and of barley roots containing different stages in the life cycle of the fungus. Immunogold labelling was used to identify the virus in sections. Labelled bundles of presumed virus particles were seen in c. 1% of zoospores liberated from plant roots and in zoospores inside zoosporangia. A few zoosporangial plasmodia had localised labelling but no bundles were seen. No virus particles were seen in sections of resting spores.     

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.     

Intracellular Localization of Jasmonate-Induced Proteins in Barley Leaves

The plant growth substance jasmonic acid and its methyl ester (JA-Me) induce a set of proteins (jasmonate-induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one-dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno-gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP-23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP-37 was detected in vacuoles and in the nucleoplasm; JIP-66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP-100 was found within the stromal fraction of chloroplasts; JIP-70 is present in the peroxisome and the nucleus; JIP-50 and JIP-6 accumulate in vacuoles. The location of JIP-66 and JIP-6 confirms their possible physiological role deduced from molecular analysis of their cDNA.     

Production and some characteristics of monoclonal antibodies against beet necrotic yellow vein virus

Four rat monoclonal antibodies (MAbs) specific for beet necrotic yellow vein virus (BNYVV) were produced. In indirect ELISA, all four MAbs reacted strongly with BNYVV infected plant leaf extracts (19 isolates from eight countries) but they did not react with beet soil-borne virus (BSBV), an unnamed rod-shaped soil-borne beet virus isolate (86 - 109) from Sweden or barley stripe mosaic virus (BSMV). However, two of the MAbs, MAFF 6 and MAFF 7 did not detect BNYVV in ELISA of infected sugar beet roots whereas MAbs MAFF 8 and MAFF 9 did detect virus in root extracts. In electro-blot immunoassay (EBIA), MAFF 6 and MAFF 7 readily detected BNYVV coat protein from leaf extracts whereas MAFF 8 and MAFF 9 reacted only weakly. None of the MAbs reacted with BSBV, 86 - 109, BSMV or plum pox virus in EBIA. MAFF 6 coated BNYVV particles which were trapped from infected leaf or root sap on to electron microscope grids by polyclonal antibodies. MAFF 6 was partially purified from tissue culture supernatant fluid by cation exchange chromatography and the preparation used to coat microtitre plates and successfully trap BNYVV in ELISA of leaf sap extracts.     

Chloroplast Protein Synthesis During Barley Leaf Growth and Senescence: Effect of Leaf Excision

Chloroplast protein synthesis was measured during the expansion,maturity and senescence of the oldest leaf of barley, Hordeumvulgare L., var. Hassan. A maximum rate of protein synthesisoccurred near the end of the expansion stage 9 d after sowing.Protein synthesis increased again at the beginning of senescenceand reached a new maximum at day 14 after sowing. Detachmentand incubation of leaves in the dark stimulated chioroplastprotein synthesis by fully expanded or by senescent leaves butnot by expanding leaves. If the detached leaves were kept inthe light, chloroplast protein synthesis was stimulated in fullyexpanded but not in senescent leaves. Short treatments (18 h)of leaf segments with growth substances in either light or indarkness, significantly changed the rate of protein synthesisshown by chloroplasts. The relationship between chloroplastprotein synthesis and leaf senescence is discussed. Key words: Hormones, light, maturity     

Structure and Photosynthetic Characteristics of Awns of Wheat and Barley

The surface and the cross section of awns of wheat and barley were examined by scanning electron microscopy,ultrastructure of cells were observed under a transmisson electron microscope and the photosynthetic rates were measured with an oxygen, electrode and infra-red CO2 analyser. The main results were as follows :The cross section of wheat awn appeared to be acutely trianglular whereas that of barley awn was obtusely triangular. There were rows of stomota on either side of epidermis in both wheat and barley awns. Under the stomatic band there were green tissues. The green cells in the awn were differentiated from the parenchyma cells . The mature green cells possessed papillae which were rich in chloroplasts and mitochondria. The tamella system in chloroplasts was well developed and contained many starch grains. There were three vascular bundles in each awn. The sheath cells near the green tissues contained chloroplasts. The photosynthate in the green cells might pass through the sheath cells and companion cells to sieve elements. The highest photosynthetic rate of the awn was seen at the flowering stage ,reaching about 20 μmol CO2·m-2·s-1. The light compensation point was 70—80 μE·m-2· s-1. The light saturation point was about 1500 μE·m-2·s-1. The CO2 compensation point was 50—60 ppm and the CO2 saturation point was about 900ppm . The photosynthetic rate and stomatal conductance were easily effected by CO2 concentration, light intensity and the duration of illumination . There was a positive correlation between the photosynthetic rate and the chloro-phyll content in the awns. The CO2-releasing rate in photorespiration of awn was about 4–5 μmol CO2·m-2·s-1.     

RT-PCR-RFLP在大麦黄矮病毒检测中的应用

The universal primer of Luteovirus was designed and synthesized.An experimental system of RT PCR RFLP was developed in barley yellow dwarf virus (BYDVs).BYDVs can be distinguished qualitatively by RT PCR method.It was seen that different serotypes of BYDVs have critical different RFLP patters when the PCR producs were digested by restriction enzyme HinfI.The RFLP patterns of 7 isolates of PAV serotype were greatly different.These results indicate there existed sequence variations among different serotypes of BYDVs and vector phenotypes of PAV serotype.There is no difference between MAV serotypes in RFLP analysis.The slight distinction in the segment of coat protein gene of BYDVs can be revealed by RT PCR RFLP system.     

Chloroplast polypeptides synthesized by leaf segments and isolated chloroplasts during senescence in barley.

Starting from senescent barley (Hordeum vulgare L. cv Hassan) leaf segments receiving light and hormone treatments affecting senescence, the plastid polypeptides synthesized by isolated chloroplasts and by leaf segments were analyzed by radiolabelling followed SDS-PAGE and fluorography. Among 20 to 30 polypeptides detected, a few were specifically synthesized (by chloroplasts and/or leaf segments) after each senescence treatment. Apparently, the polypeptides labelled in assays with isolated chloroplasts are truly synthesized in vivo, because most of them were also labelled in assays with leaf segments. The comparison of polypeptide profiles, for every senescence treatment, after labelling with isolated chloroplasts or leaf segments, suggests that most plastid polypeptides synthesized during senescence are coded in plastid DNA.