Rapid Detection of Potato Spindle Tuber Viroid (PSTV) by Dot Blot Hybridization

A PSTV probe of 350 bases, prepared in our laboratory, was used to detect picogram quantities of viroids by a simplified, improved, dot blot, hybridization technique in crude potato and tomato extracts. Optimum conditions for certification of PSTV-free potato plants were established, involving formamide concentration, washing stringencies, exposure time during autoradiography and method of probe radiolabelling. Ten pg of purified PSTV in water and 50 pg of purified PSTV added to healthy plant extract were detected. Also hybridization signals could be detected from as little as 0.075 mg of infected plant tissue.     

用聚合酶链式反应（PCR）检测马铃薯纺锤块茎类病毒

用DNA合成仪合成两个马铃薯纺锤块茎类病毒(Potato spindle tuber viroid, PSTVd)特异性引物,从感病的马铃薯块茎组织的核酸抽提液中,用反转录酶合成PSTVd eDNA,然后用PCR法进行扩增,扩增产物用电泳检测,建立了用PCR法检测PSTVd的新方法。结果表明,该方法特异性强,灵敏度可达0.15pg,比现有其它检测方法高,而且样品用量少。     

Scion on a Stock Producing siRNAs of Potato Spindle Tuber Viroid (PSTVd) Attenuates Accumulation of the Viroid

Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.     

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。     

Survival and Transmission of Potato Virus Y,Pepino Mosaic Virus,and Potato Spindle Tuber Viroid in Water

Hydroponic systems and intensive irrigation are used widely in horticulture and thus have the potential for rapid spread of water-transmissible plant pathogens. Numerous plant viruses have been reported to occur in aqueous environments, although information on their survival and transmission is minimal, due mainly to the lack of effective detection methods and to the complexity of the required transmission experiments. We have assessed the role of water as a source of plant infection using three mechanically transmissible plant pathogens that constitute a serious threat to tomato and potato production: pepino mosaic virus (PepMV), potato virus Y (PVY), and potato spindle tuber viroid (PSTVd). PepMV remains infectious in water at 20 ± 4°C for up to 3 weeks, PVY (NTN strain) for up to 1 week, and PSTVd for up to 7 weeks. Experiments using a hydroponic system show that PepMV (Ch2 genotype) and PVY (NTN strain) can be released from plant roots into the nutrient solution and can infect healthy plants through their roots, ultimately spreading to the green parts, where they can be detected after a few months. In addition, tubers developed on plants grown in substrate watered with PSTVd-infested water were confirmed to be the source of viroid infection. Our data indicate that although well-known pathways of virus spread are more rapid than water-mediated infection, like insect or mechanical transmission through leaves, water is a route that provides a significant bridge for rapid virus/viroid spread. Consequently, water should be taken into account in future epidemiology and risk assessment studies.     

黄瓜花叶病毒卫星RNA与马铃薯纺锤形块茎类病毒间序列同源性与碱基配对

黄瓜花叶病毒卫星ＲＮＡ与马铃薯纺锤形块茎类病毒间序列同源性与碱基配对田波（中国科学院微生物研究所,北京１０００８０）Ｇ．ＳｔｅｇｅｒＤ．Ｒｉｅｓｎｅｒ（ＩｎｓｔｉｔｕｔｆｕｒＰｈｙｓｉｋａｌｉｓｃｈｅＢｉｏｌｏｇｉｅ,ＵｎｉｖｒｓｉｔａｔＤｕｓｓｅｌ...     

Effects of Potato Spindle Tuber Viroid Infection on Phytohormone and Antioxidant Responses in Symptomless Solanum laxum Plants

Journal of Plant Growth Regulation - To better understand the role of phytohormones and antioxidative responses in plant-viroid interaction, changes in endogenous phytohormone contents and...     

特异切割马铃薯纺锤形块茎类病毒负链的多价核酶的构建和体外活性测定

根据锤头型核酶的作用模式 ,设计、合成并克隆了特异切割马铃薯纺锤形块茎类病毒 (PSTVd)负链RNA不同区域位点的双价和三价锤头型核酶基因。通过体外转录 ,将PSTVd负链RNA分别与双价和三价核酶混合 ,37℃温育 2h。结果表明 ,双价核酶和三价核酶均表现出较高的切割活性 ,其中双价核酶处理的切割产物的大小与理论值相符合。三价核酶虽表现出较高的切割活性 ,但只是其中一价核酶在起作用。讨论了二价和三价核酶的应用前景。     

Biotransformation of Paclitaxel (Taxol) by the Cell Suspension Cultures of Rauwolfia serpentina

The biotransformation of paclitaxel (TaxolⓇ) by the cell suspension cultures of Rauwolfia serpentina (L.) Benth. et Kurz. were investigated. Three Paclitaxel-based intracellular metabolites were detected from the cell filter cake and were, by high field ¹H-NMR and MS data, identified as 10-deacetyltaxol, baccatin Ⅲ, and 10-deacetylbaccatin Ⅲ. No glucosidated or hydroxylated derivatives were checked out in this incubation experiment.     

条件培养液对红豆杉细胞Paclitaxel生产的促进作用

在两步法红豆杉（Taxus chinensis)细胞悬浮培养体系的生产阶段,加入从生长阶段悬浮培养物中制得的条件培养液（conditioned Medium,CM)既能促进细胞的生长,又能提高紫杉醇（paclitaxel)的产率,解决了生产培养时,细胞生长受抑制的问题,特别是,取自生长12天的细胞悬浮培养物的CM按体积分数为25％添加到新鲜生产培养基中时,可使细胞紫杉醇最高产量达28．5mg/L,细胞干重达32．3g/L,分别是对照的2．4倍和2．2倍,对CM中的蔗糖,果糖,NO3－和PO4－3等的含量的进行了分析。     

蛇根木细胞悬浮培养进行紫杉醇的生物转化

通过理化及光谱学方法,从蛇根木(Rautwolfia serpentina(L.)Benth.et Kurz.)悬浮细胞内分离到3个紫杉醇同系物,高分辨1H-NMR和MS结构分析表明,它们分别为10-脱乙酰紫杉醇、baccatinⅢ和10-deacetylbaccatinⅢ.本实验未检测出紫杉醇的苷化或羟基化衍生物.     

大麦胚性细胞悬浮系的建立及其生长特性

由春大麦品种“如车”种胚诱导的松脆型胚性愈伤组织经2个月的悬浮培养,成功建立分散性好、生长速度快的胚性细胞悬浮系。该系细胞直径为1-3mm,由富含淀粉粒的胚性薄壁细胞构成。经不同浓度2,4-D实验,发现2mg/L最适合该细胞系的生长。文中对成功建立大麦胚性细胞悬浮系的关键问题进行了讨论。     

紫花曼陀罗悬浮培养细胞转化对羟基苯甲醛生产天麻素

利用紫花曼陀罗细胞悬浮培养转化外源对羟基苯甲醛合成天麻素,并应用多种色谱技术进行分离纯化,根据转化产物的理化性质和光谱数据分析鉴定结构。实验表明,紫花曼陀罗细胞成功将对羟基苯甲醛转化为天麻素(Ⅱ),同时也得到了由对羟基苯甲醛生成天麻素的转化中间体对羟基苯甲醇(Ⅰ)。在培养基中添加0.1mg/L的水杨酸能显著提高细胞对外源对羟基苯甲醛的糖基化率,而保持气升式发酵罐(25-L)罐内压力为低压(0.001MPa)也能提高细胞对外源对羟基苯甲醛的糖基化率。实验证明,紫花曼陀罗细胞悬浮培养能有效转化对羟基苯甲醛合成天麻素。     

长春花悬浮培养细胞对天麻素的生物转化

应用长春花(Catharanthus roseus (L.) G. Don)悬浮细胞培养体系对天麻素进行了生物转化反应研究.经过8 d培养形成一个转化产物,应用光谱方法鉴定转化产物的结构为对羟基苯甲醇,为天麻素水解后形成的甙元.     

Glucosylation of Capsaicin by Cell Suspension Cultures of Coffea arabica

Capsaicin was converted into the corresponding glucoside when administered to cell suspension cultures of Coffea arabica cultured in a modified Murashige and Skoog’s medium with 5 μM 2,4-dichlorophenoxyacetic acid and 0.5 μM kinetin. The glucoside was identified as capsaicin-β-D-glucopyranoside by FAB-MS, ¹H-NMR, and hydrolysis with α- and β-glucosidases. The pungency of the glucoside was approximately 1/100 of that of capsaicin.