柑桔裂皮类病毒感染——爪哇三七的研究

爪哇三七(Gynura aurantiaca)是柑桔裂皮类病毒(CEV)的敏感鉴定植物之一,也是CEV能得到大量增殖的寄主植物。本文报告了采用反向双相聚丙烯酰胺凝胶电泳银染色法和分子杂交技术,对CEV感染爪哇三七的若干特性的研究结果表明:感染了CEV的爪哇三七,无论是否表明症状,都比健株要多一条CEV-RNA带;感染了CEV的爪哇三七核酸提取物与CEV-cDNA探针能产生杂交点,而健株的核酸提取物则不能。以感病爪哇三七植株上发病的芽,再进行剪插,其病症的表现与光照有关,但经电泳检测CEV-RNA带的存在与病症表现无关。生物鉴定是对病毒及类病毒进行鉴定的经典方法。人们发现芸香科(Rutaceac)的香橼(Citron)等对柑桔裂皮病类病毒(CEV)敏感。此外,菊科(Compositae)的爪哇三七(Gynura aurantiaca)是对CEV敏感的草木植物之一。国内从部分地区田间柑桔裂皮病症状的调查和以香橼为指示植物鉴定CEV发生情况等研究都已有报道,但要对CEV的理化性质和分子生物学性质作深入研究,搞清楚CEV的致病机理及复制方式,并找到防治CEV的有效对策,首先需要大量高纯度的CEV-RNA,而直接从柑桔叶片中提取CEV则相当困难。爪哇三七不仅是CEV的敏感鉴定植物,可用于柑桔裂皮病的早期生物诊断,同时也是CEV大量增殖的理想寄主。为此,已有学者从CEV影响爪哇三七插条生根,使植株整体变化到CEV引起爪哇三七细胞病变,以及CEV引起爪哇三七蛋白质的变化等都作了细致的研究和报道。近年来,类病毒的研究方法又有新的进展,除了用简便的聚丙烯酰胺凝胶电泳(PAGE)甲苯胺兰染色法外,又采用了灵敏的反向双相PAGE银染色法来检测类病毒。我们采用双相PAGE银染色法和分子杂交技术,进一步考察了CEV感染爪哇三七的若干特性,其结果报告如下     

柑桔裂皮病类病毒一爪哇三七悬浮细胞的生长特性

爪哇三七(Gvnura aurantlaca Dc)是柑桔裂皮病类病毒(Citrus Exocortis Viroid简称CEV)敏惑的指示植物。用CEV感染的柑桔叶研磨汁液机械接种健康爪哇三七植株,以此作为CEV感染的外植体采源。在MS和B,琼脂培养基上诱导和继代培养健康、CEV感染的爪哇三七叶片的愈伤组织,在MS、B,液体培养基中建立健康、CEX’感染的爪哇三七悬浮细胞系统。从愈伤组织和悬浮细胞培养物中抽提核酸,经5％双向聚丙烯酰胺凝胶电泳、银染分析：在健康爪哇三七愈伤组织和悬浮细晦中不出现特异的CEV电泳带,而CEV感染的爪哇三七的所有培养物中都含有类病毒。cEV可在有丝分裂征盛的爪哇三七悬浮细胞中复制。散伤组织和悬浮细胞很容易继代培养和保存。     

柑桔裂皮病类病毒在爪哇三七不同器官中的分布

采用生物学方法,正反两相聚丙烯酰胺凝胶电泳及分子杂交技术,对感染了柑桔裂皮类病毒(CEV)且发病的爪哇三七根、茎、叶、花及种子各部分器官的带毒情况进行了检测分析,研究了CEV在宿主体内的分布。结果表明:CEV在惑染爪哇三七体内呈周身分布,但分布是不均一的。     

感染柑桔裂皮病类病毒的悬浮细胞的一些特性

爪哇三七(Gynura aurantiaca D.C.)是柑桔裂皮病类病毒(Citrus Exocortis Vi-roid,简称CEV)敏感的指示植物。我们建立了健康和CEV感染的爪哇三七悬浮细胞培养的体外系统。绘制了悬浮细胞培养的生长曲线、pH曲线和温度曲线。CEV可以在悬浮细胞中复制。对继代培养中CEV和寄主核酸的连续测定表明CEV的扩增阻遏了寄主核酸的复制。     

一种有效的分离制备柑桔裂皮病类病毒的方法

类病毒分子量小,在寄主体内含量低,不易提纯。一般提纯方法需经过酚抽提,去糖、透析、氯化锂处理、DNase消化和纤维素层析,由于步骤复杂,所以回收率较低,同时所得样品纯度不高,难以满足各种需要。本文报道一种柑桔裂皮病类病毒(CEV)的分离制备方法,采用苯酚-氯仿抽提核酸,核酸经LiCl处理后,进行纤维素(CF-11)层析,然后经PAGE纯化CEV。该方法具有操作简便,所使用的设备简单,纯化CEV效果好等优点,是柑桔裂皮病类病毒早期诊断,序列分析,分子克隆和防治等研究必不可少的基础工作。     

几种化学药物对柑桔裂皮病类病毒感染和复制的影响

经柑桔裂皮病类病毒(Citrus exocortis viroid,简称CEV)感染后的指示植物爪哇三七(Cynura aurantiaca),分别施用不同浓度的赤霉素(5ppm,10ppm,50ppm,100ppm,200ppm)、萘(100ppm,300ppm)、5-氟尿嘧啶(0.5mg/L,1mg/L,2mg/L)溶液,其相对感染指数(Relative infectivity index)均有程度不同的改变。抽提叶片核酸,经5％双向聚丙烯酰胺凝胶电泳(PAGE)-银染色法结合凝胶扫描技术确定CEV浓度,辅以点杂交方法定性,观察到施用赤霉素组CEV浓度增高,施用萘组和5-氟尿嘧啶组CEV浓度均降低。用无性繁殖的柑桔苗作材料,CEV感染后分别施用50ppm赤霉素、300ppm萘、1mg/L 5-氟尿嘧啶溶液,用相同的分析方法,得到了与爪哇三七一致的结果。讨论了这三种药物影响CEV复制的意义。     

爪哇三七组织培养类病毒的初步研究

用含裂皮病类病毒(Citrus exoeortis viroid,简称CEV)的古巴花叶橙、冰糖橙的叶汁和核酸粗提物感染爪哇三七(Gynura aurantiaca),取其腋芽和嫩叶作外植体,经消毒处理,接种在稍加改良的B5和MS半固体培养基上,置于24℃～27℃遮光培养,经2～3周诱导形成愈伤组织,质地有的松散易碎似白木耳状,有的坚硬灰黑似多瘤状,经双向凝胶电泳-银染法和点杂交法检测,证实用CEV感染的嫩叶和腋芽作外植体,不仅能诱导形成愈伤组织,而且CEV还可能在愈伤组织中复制,结果表明,已初步建立了爪哇三七的组织培养和研究类病毒的离体培养系统。     

爪哇三七组织培养植株再生

本文报道了爪哇三七通过组织培养再生植株的方法。爪哇三七的叶片,叶柄或茎段外植体分别接种于6种分化培养基上,均难以直接分化。在附加1mgL~(12),4-D和0.1mgL~(-1)KT的B_5培养基上的叶片形成无色疏松的愈伤组织转接于MS附加1mgL~(-1)BAP和0.1mg L~(-1)NAA培养基上,5天后开始形成绿色球胚状结构,继而形成不定芽或丛生芽。这些不定芽或丛生芽在锈根培养基中迅速长成根系发达的完整植株。此外,本试验比较了不同浓度及不同组合的激素对爪哇三七外植体的脱分化、再分化的影响。并讨论了此项工作对柑桔裂皮病类病毒(简称CEV)的复制及致病机理研究的意义。     

柑桔根繁能脱除裂皮病类病毒吗?

柑桔裂皮病类病毒(Citrus Exocortis Vitold,CEV)的传播和流行,对柑桔的生产带来了严重的损失。已证明特CEV的亲本可通过嫁接、插枝这两种营养繁殖的方式将CEV传播给子代。所以,CEV脱毒方法就成为生产上急待解决的问题。据报道,采用热处理、茎尖微芽嫁接和选育珠心苗等技术可获得无毒植株。嫁接是一种常用的育苗方法,然     

柑桔裂皮病类病毒感染爪哇三七叶片原生质体

柑桔裂皮病类病毒(Citrile Exocortis Viroid,简称CEV)是一种严重影响柑桔生产的单链闭环RNA致病因子,近年来人们已对它的理化特性、检测方法以及离体培养系统进行了研究。由于利用原生质体出利用整个植物或愈伤组织来研究CEV复制和致病机理更为优越,所以建立一个适合CEV感染的原生质体体系是人们关心的重要课题。     

Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton

Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome-encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves.     

The role as inoculum sources of Xanthomonas citri pv. citri surviving on the infected Satsuma mandarin fruits

Importing citrus fruits infected by Asiatic citrus canker caused by Xanthomonas citri pv. citri (Xcc) can act as an inoculum source for the disease epidemic in citrus canker-free countries. In this study, the pathogenicity of the causal agent of Asiatic citrus canker surviving on infected Satsuma mandarin fruits was evaluated. The washing solution of infected Satsuma mandarin fruits did not cause lesion formation on the citrus leaves. However, a typical citrus canker lesion was formed on the leaves after inoculation with higher concentrations of the inoculum from the washing solution (washing solution II). It indicated that the pathogenicity of the citrus canker surviving on the symptomatic Satsuma mandarin fruits was not changed. Scanning electron microscopic observation showed that the numbers of bacterial cells on the leaves of Satsuma mandarin which inoculated with the washing solution directly (washing solution I) was less compared to those of leaves inoculated with the washing solution II. This result spports that the pathogenicity of Xcc surviving on Satsuma mandarin fruits may not be changed but that the sucessful infection of citrus caker may depend on the concentration of the inoculum.     

Proteins associated with salt adaptation in citrus and tomato cells: Involvement of 26 kDa polypeptides

Stress-induced changes in proteins of molecular weights of 25 and 27 kDa are reported for NaCl-adapted cells of Shamouti orange ( Citrus sinensis L. Osbeck) and the cultivated VFNT cherry tomato ( Lysopersicon esculentum Mill.), respectively. Salt-adapted cell lines of both species grew much faster in the presence of salt than non-adapted cells and their growth rate was similar to that of non-adapted cells in the absence of salt. Sodium dodecyl sulfate-extractable citrus and tomato polypeptides were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). The enhancement of a citrus 25 kDa protein associated with salt tolerance was actually resolved only on two-dimensional PAGE. It is a constitutive protein, i.e. it was present in salt-tolerant ceils whether grown in the presence or absence of salt. The NaCl-induced changes of the tomato 27 kDa protein could be resolved on one-dimensional PAGE. This protein was first markedly suppressed by a salt shock and the time required for its recovery was dependent on the degree of salt tolerance. Prolonged adaptation resulted in an increase of this protein. Protein profiles of citrus and tomato cells grown in the absence and presence of salt revealed that while in citrus the level of most proteins was suppressed in the presence of salt, the opposite was true for tomato proteins under similar conditions. Comparison between the changes in citrus and tomato and results already published for tobacco show that many salt-induced changes in proteins are species-specific and that no obvious similarities exist.     

Saturating Irradiance-Induced Photoinhibition without Monomerisation of Photosystem 2 Dimer in Soybean Leaves

The oligomeric state of photosystem 2 (PS2) complex in soybean leaves treated with saturating irradiance was studied by non-denaturing polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography. PS2 dimers resolved by non-denaturing PAGE accounted for about 75 % of total PS2 complex and there was no significant difference in the ratio of PS2 dimer to monomer between samples from saturating irradiance-treated and fully dark-adapted leaves. Furthermore, BBY particles were resolved into four chlorophyll-enriched fractions by gel filtration chromatography. From their molecular masses and protein components, these fractions were deduced to be PS2 dimer, PS2 monomer, oligomeric light-harvesting complex 2 (LHC2), and monomeric LHC2. Also, no change in the proportion of PS2 dimer in total PS2 was observed in the granal region of thylakoid membranes from soybean leaves after saturating irradiation. Hence the dimer is the predominant natural form of PS2 in vivo and no monomerisation of PS2 dimer occurs during saturating irradiance-induced photoinhibition in soybean leaves.     

双向电泳分析鸢尾绿白嵌合叶片的蛋白质

利用双向聚丙烯酰胺凝胶电泳对鸢尾（Iris japonica)绿白嵌合叶片的蛋白质进行分离,并初步鉴定了蛋白质的相对分子量和等电点。每个电泳图谱共检测到400余个蛋白点,其中至少13个蛋白的表达变化明显;结果表明,嵌合叶片的绿色与白色叶组织具有明显不同的蛋白质双向电泳图谱。与数据库中拟南芥双向电泳图谱相比较,发现Rubisco大亚基,标记为W和T蛋白的表达变化与产生绿白嵌合叶片的表型密切相关。