大麦黄花叶病毒侵染的大麦叶肉细胞超微结构研究

在自然感染大麦黄花叶病毒的大麦叶肉细胞中可见线条状和杆状的病毒粒体以及风轮状内含体。这些病毒的长度一般为480—920nm,宽为lo—20nm。此外,还观察到一种由许多病毒组成的堆束状结构,这种病毒的直径为13nm 左右,长度可达2000nm 以上。感病叶肉细胞的超微结构变化是相当明显的。在病害严重的细胞中,细胞基质丧失严重;叶绿体膜系统破坏;线粒体的嵴和基质减少;内质网膨大或断裂,小泡大量出现,病毒粒体的一端往往与内质网联结在一起,特征性膜性网络结构在感染的细胞质中形成。细胞核和细胞膜也发生了变化。     

大麦黄花叶病毒(BaYMV)的提纯和生化性质的研究

大麦黄花叶病是由禾谷多粘菌(polymyxa graminis)传布的一类麦类病毒病。是上海市效区及长江中下游及沿海大麦产区的重要病害。大麦黄花叶病毒(BaYMV)在国内外虽已有不少研究,但对国内分离株的生化性质尚未有详细报道。本文报道以上海郊区的BaYMV为研究材料,成功地建立了一套分离提纯的方法,并在此基础上,研究了该病毒的结构蛋白和核酸性质,并和国外的一些研究结果进行了比较,从而为今后应用生物技术防治大麦黄花叶病毒打下了基础。     

应用单链构象多态性—聚合酶链反应研究我国大麦黄花叶病毒株系的分化

根据致病性差异,我国大麦黄花叶病毒（ＢａＹＭＶ）存在若干不同株系,血清学和核酸杂交技术不能区分这些株系。但还由于致病性不同,各株系的核酸序列同必定存在差异。最近建立的单链构象多态性－聚合酶链反应（ＳＳＣＰ－ＰＣＲ）技术能灵敏而有效地诊断和区分核酸序列间的差异,从而进一步证实我国ＢａＹＭＶ不同株系的分化。     

中国大麦黄花叶病毒分离物的分子变异

１３个供试的中国和英国大麦黄花叶病毒（ＢａＹＭＶ）分离物,经ＲＮＡ１和ＲＮＡ２全基因组不同区域ＤＮＡ片段背地里单构象多态分析（ＳＳＣＰ）,外壳蛋白基因和ＲＮＡ２ ７０ｋＤ基因５端７０５碱基序列分析,以及此７－５碱基ＤＮＡ片段限制性内切酶图谱分析结果,它们的ＲＮＡ１和ＲＮＡ２彼此无一相同,其中ＲＮＡ２变异比ＲＮＡ１更大。由于变异十分复杂,且没有规律性,因而当前通用的分子生物学技术尚不能简单地ＢａＹＭ     

大麦黄花叶病毒属成员UTR系统进化树分析及编码蛋白跨膜结构推测

对大麦黄花叶病毒属成员的同源性和系统进化树分析表明,同一病毒RNA1和2基因组5′-UTR区域在进化上的相关性高于不同病毒同一RNA组分间的关系,而3′-UTR则相反.蛋白质二级结构分析显示RNA1编码的P3和14K蛋白存在跨膜结构.BaMMV ALS1分离物P3蛋白跨膜结构在大肠杆菌中原核表达时有致死作用.类比Potyviridae科其它属成员功能,此2个蛋白可能与膜附着功能相关.RNA2编码的P2蛋白存在两个结构保守的跨膜区域,一些摩擦接种保存的大麦和性花叶病毒(BaMMV)分离物和燕麦花叶病毒(OMV) P2蛋白此两个结构(或一个)缺失后,不能由介体禾谷多粘菌(P. graminis)传播,揭示P2蛋白跨膜结构与禾谷多粘菌传播能力相关.     

应用单链构象多态性－聚合酶链反应研究我国大麦黄花叶病毒株系的分化

根据致病性差异,我国大麦黄花叶病毒（ＢａＹＭＶ）存在若干不同株系,血清学和核酸杂交等技术不能区分这些株系。但还由于致病性不同,各株系的核酸序列间必定存在差异。最近建立的单链构象多态性。聚合酶链反应（ＳＳＣＰ－ＰＣＲ）技术能灵敏而有效地诊断和区分核酸序列间的差异,从而进一步证实我国ＢａＹＭＶ不同株系的分化。     

大麦黄花叶病毒属成员UTR系统进化树分析及编码蛋白跨膜结构推测

对大麦黄花叶病毒属成员的同源性和系统进化树分析表明 ,同一病毒RNA1和 2基因组 5′ UTR区域在进化上的相关性高于不同病毒同一RNA组分间的关系 ,而 3′ UTR则相反。蛋白质二级结构分析显示RNA1编码的P3和 14K蛋白存在跨膜结构。BaMMVALS1分离物P3蛋白跨膜结构在大肠杆菌中原核表达时有致死作用。类比Potyviridae科其它属成员功能 ,此 2个蛋白可能与膜附着功能相关。RNA2编码的P2蛋白存在两个结构保守的跨膜区域 ,一些摩擦接种保存的大麦和性花叶病毒 (BaMMV)分离物和燕麦花叶病毒 (OMV)P2蛋白此两个结构(或一个 )缺失后 ,不能由介体禾谷多粘菌 (P .graminis)传播 ,揭示P2蛋白跨膜结构与禾谷多粘菌传播能力相关     

不同抗性的榨菜在芜菁花叶病毒感染后细胞超微结构的研究

超薄切片观察表明,在三种感染芜菁花叶病毒的榨菜叶肉细胞内均发现有各种形状的内含体和病毒粒子的存在。内含体形状有风轮状、束状、管状、环状和板状聚集体,且不同抗性的榨菜品种细胞内所含的内含体数量、种类及所占的比例亦不同,抗性较弱的９０－１４９榨菜品种叶肉细胞内内含体数量较多,以管状及环状内含体所占比例为多,束状及风轮状内含体所占比例较少;抗性较强的９０－１３９榨菜品种叶肉细胞内内含体数量较少,且以束状及风轮状内含体所占比例较多,环状及管状内含体所占比例较少。而抗性中等的９０－１４６榨菜品种叶肉细胞内内含体数量及所占比例介于上述两者之间。病毒粒子也以易感病的９０－１４９榨菜叶肉细胞内含量最多,均以束状或拟晶格状存在。此外感病初期细胞内线粒体及粗面内质网数量有不同程度的增多,且以易感病的品种叶肉细胞内这些细胞器增加明显,但健康植株内以抗性较强的品种含这些细胞器较多。随着病毒症状的发展,叶绿体畸形,肿大,基质消失,外膜破裂直至解体。     

应用F(ab'''')_2~-酶联吸附分析法检测大麦黄花叶病毒

F(ab′)_2酶联免疫吸附分析法(F(ab′)_2-ELISA)成功地用于大麦黄花叶病毒(BaYMV)的常规检测和诊断.其步骤是先用稀释1000—4000倍的抗血清F(ab′)_2包被反应板,加待测样品和稀释1000倍的同种抗血清或IgG,然后再加A蛋白碱性磷酸酯酶和底物,测定OD值。比较试验表明,ELISA稀释缓冲液加入1％小牛血清或1％全脂奶粉,BaYMV的测检灵敏度可提高达2.5—5.0ng/ml,病叶汁液检测终浓度为稀释1600—3200倍。我国BaYMV分离物与英国分离物的血清学性质完全一致。BaYMV在大麦病株中以叶部含量较高,茎中含量次之,根部测不出病毒。检测和诊断田间样品,即使有的样品已不新鲜,也均能得到满意的结果。此方法也成功地用于大麦温和花叶病毒(BaMMV)、小麦黄花叶病毒(WYMV)、燕麦花叶病毒(OMV)和燕麦金色条纹病毒(OGSV)等禾谷多粘菌传麦毒的检测,这S种病毒的血清学关系研究表明,除BaYMV和WYMV之间具有血清学关系以外,其余彼此均不反应。     

杉木小孢子核内双膜包绕的内含体(简报)

杉木小孢子的发育已进行了系统的电子显微镜研究。在研究中我们发现小孢子发育到一定阶段,细胞核内出现一种双膜包绕的内含体。这种有膜内含体无论在结构上,发生的普遍性和阶段性上都与已有的报道不同,是首次发现。研究材料为杉木(Cunninghamia lanceola-     

Strategies for Pyramiding Resistance Genes Against the Barley Yellow Mosaic Virus Complex (BaMMV,BaYMV, BaYMV-2)

Barley Yellow Mosaic Virus disease caused by different strains of BaYMV and BaMMV is a major threat to winter barley cultivation in Europe. Pyramiding of resistance genes may be considered as a promising strategy to avoid the selection of new virus strains and to create more durable resistances. However, this goal cannot be achieved by phenotypic selection due to the lack of differentiating virus strains. For pyramiding of resistance genes rym4, rym5, rym9 and rym11, located on chromosomes 3H and 4H of barley two different strategies have been developed. These strategies are based on doubled haploid lines (DHs) and marker assisted selection procedures. On the one hand F₁ derived DH-plants of single crosses were screened by molecular markers for genotypes being homozygous recessive for both resistance genes. These genotypes were crossed to lines carrying one resistance gene in common and an additional third gene, leading to a DH-population of which 25% carry three resistance genes, 50% have two resistance genes and 25% possess a single resistance gene homozygous recessively. Alternatively, F₁ plants having one resistance gene in common were directly inter-crossed [e.g. (rym4 × rym9) × (rym4 × rym11)] and about 100 seeds were produced per combination. Within these complex cross progenies plants were identified by markers being homozygous at the common resistance locus and heterozygous at the others. From such plants, theoretically present at a frequency of 6.25%, DH-lines were produced, which were screened for the presence of genotypes carrying three or two recessive resistance genes in a homozygous state. Besides DH-plants carrying all possible two-gene combinations, 20 DH-plants out of 107 analysed carrying rym4, rym9, and rym11 and 27 out of 187 tested carrying rym5, rym9, and rym11 homozygously have been detected using the second strategy which is faster but needs co-dominant markers, because in contrast to the first strategy marker selection is carried out on heterozygous genotypes.     

Ultrastructural Studies of Barley Mesophyll Cella lnfected with Barley Yellow Mosaic Viruses

lexuous filamentous, rod-shaped particles, and laminated, pinwheel inclusions were observed in the mesophyll cells of the barley plants naturally infected with barley yellow mosaic viruses. These virus particles had a length of 480–920 nm and a width of 10–20 nm. In addition, bundles of filamentous structures which consisted of many particles with more 2000 nm in length were found in the leaves of the infected barley plants. The ultrastructural alterations of the infected mesophyll cells were rather conspicuous. The cytoplasmic matrix was lost seriously, and the chloroplast membrane system was destroyed. The cristae and matrix of the mitochondrium were decreased and some of them became vacuoles. The endoplasmic reticulum (ER) expanded teristic membranous network structures occurred in the cytoplasm of infected cells. The virus particles were often associated at one end with ER and with the membranes of network structures. The nucleus, membrane and wall of ceils also had somewhat variation.     

菜豆黄色花叶病毒(BYMV)—新疆苜蓿分离株的鉴定

从新疆苜蓿黄斑花叶病株上分离到病毒分离物M-4,该分离物能引起多种豆科值物系统花叶,并在藜科植物上产生局部褪绿斑,易经汁液摩擦接种和蚜虫传毒,不经菜豆种子传毒。病毒致死温度60—65℃,体外保毒期4—5天,稀释限点10~(-3)—10~(-4)。病毒粒体线状,长约660—740nm,宽15nm;在感病的寄主叶片细胞中,电镜观察到风轮状、带状和环状内含体。免疫电镜法测定,该分离物与菜豆黄色花叶病毒(BYMV)抗血清有血清反应。经SDS-聚丙烯酰胺凝胶电泳和氨基酸自动分析仪分析分别测得该病毒的衣壳蛋白亚基分子量为16,200道尔顿,氨基酸残基数128个。鉴定结果认为,分离物M-4是BYMV的一个株系。     

Molecular mapping of novel resistance genes against Barley Mild Mosaic Virus (BaMMV)

 In the present study three novel genes from barley accessions 10247 (ym8), Bulgarian 347 (ym9), and Russia 57 (ym11), which confer resistance to Barley Mild Mosaic Virus (BaMMV), were mapped using molecular markers. Bulked segregant analysis of four progenies segregating for resistance to BaMMV was followed by fine-scale mapping of the resistance genes using individual F₂ or BC₁F₂ plants. The resistance genes are inherited recessively and are located on the long arm of barley chromosome 4HL. A series of closely linked molecular markers are available for marker-assisted breeding programs. A marker (MWG2134) linked with resistance gene ym11 from Russia 57 was identified, which is diagnostic for the resistance gene. Received: 25 July 1997 / Accepted: 22 August 1997     

应用免疫胶体金技术定位感病大麦细胞中的大麦和性花叶病毒

本文报道免疫胶体金标记技术的建立,并用此技术定位大麦叶和根组织超薄切片中大麦和性花叶病毒(BaMMV)。在感染病毒的大麦叶和根细胞中,病毒束、游离病毒颗粒和病毒外壳蛋白多分布于细胞质丰富的细胞中,且以液泡和叶绿体(仅叶组织)周围较多。在细胞器已解体的病根表皮细胞中,有时也可检测到大量游离病毒粒子。少数风轮体或板状集结体上也存在病毒或病毒外壳蛋白。细胞核、叶绿体、线粒体、细胞膜以及其他细胞器上都未见有特异性金颗粒标记。     

小麦土传病毒在感病小麦细胞中的分布及小麦梭条斑花叶病毒RNA组分

四川雅安、陕西长安的土传小麦病毒病由小麦梭条斑花叶病毒(WSSMV)引起,而浙江安吉、新昌、江苏宜兴的病害则由WSSMV和土传小麦花叶病毒(SBWMV)所致。WSSMV和SBWMV可以同时复合侵染同一株小麦,但在病细胞中二者彼此独立分布。我国WSSMV RNA有2个基因组,分子量分别为2.6×10~6和1.5×10~6,与日本小麦黄花叶病毒(WYMV)一致。     

禾谷多粘菌对小麦梭条斑花叶病毒的传播及其土壤侵染潜力的测定

从大田侵染小麦梭条斑花叶病毒的小麦病根中挑取禾谷多粘菌休眠孢子堆,接种受侵染小麦品种扬麦４号,经砂培养纯化,获得５个禾谷多粘菌分离物,但都为无毒。无毒多粘菌休眠孢子堆接种表现ＷＳＳＭＶ症状的小麦,经培养可饲获病毒,并可经接咱后将病毒传播给无病小麦,供试的４个大小麦禾谷多粘菌分离物都可对大小进行交叉侵染,产生同样数量的游动孢子产量。供试５个病土和２个无病土样品,都具有强大持多粘菌侵染潜力,即使稀释放