Detection of strains of potato virus S by nucleic acid spot hybridization (NASH)

Summary Two complementary DNA (cDNA) clones (0.9 kb and 1.6 kb) reacting to the ordinary strain of potato virus S (PVS) were compared with single-stranded randomly primed cDNA (prepared to total genomic RNA) as probes for various strains of PVS, using the technique of nucleic acid spot hybridization (NASH). The cDNA clones detected 11 PVS isolates well, including both Andean and ordinary strains, while one of each strain was detected weakly. As little as 5 femtograms of PVS RNA could be detected. A low cross hybridization was observed with cDNA probes to PVS with potato virus M and carnation latent virus both members of the carlavirus group. No cross homology was detected with seven other potato viruses. NASH was as sensitive and specific as enzyme-linked immunosorbent assay (ELISA), with no pretreatments of crude sap being required before spotting on nitrocellulose filters. Cloned cDNA probes would be suitable for mass screening of PVS.     

Some observations on the nucleic acids content in the different buds of etiolated and green Phaseolus plants

Summary In this paper are presented some observations about the nucleic acids content in the different buds of eight days old green and etiolatedPhaseolus plants.Obvious differences of nucleic acids content and typical changes in RNA/DNA ratio in the bud of etiolated and green plants were found. The probable role of the differences in nucleic acids on the histological and morphological differences that take place with etiolation is discussed.     

Detection on polyacrylamide gel of a diagnostic nucleic acid from tissue infected with potato spindle tuber viroid

A diagnostic procedure for potato spindle tuber disease is described which allows detection of the causal viroid in small amounts of potato and tomato tissue. The method involves extraction of cellular nucleic acids, their separation by polyacrylamide gel electrophoresis and staining with toluidine blue O. The procedure has been used to index greenhouse and field potatoes for mild and severe strains of the pathogen     

Multiplex detection ofPotato virus S, Potato virus X, andPotato virus Y by non-radioactive nucleic acid spot hybridization in potato tissue culture plantlets

Potato plantlets initiated into tissue culture must be tested for numerous viruses prior to propagation for seed potato production. Ideally, one plantlet is tested for all pathogens of concern and, if found pathogen-free, this plantlet is propagated for production of seed potatoes. Commercially available ELISA kits are generally used for the pathogen tests, but the commercial kits have some limitations. For example, the protocols differ for different viruses, so multiple extractions must be completed, increasing the time and expense of testing. This is a significant problem with tissue culture plantlets, for which there is limited material available to test and an ever-increasing number of pathogens that must be tested for, including viruses in the potyvirus, carlavirus, potexvirus, luteovirus, pomovirus, tobravirus, tospovirus, alfamovirus, and tymovirus groups. We have optimized a non-radioactive nucleic acid hybridization (NASH) assay for the simultaneous detection of carlavirusPotato virus S (PVS), potexvirusPotato virus X (PVX) and potyvirusPotato virus Y (PVY) in potato tissue culture plantlets. This assay requires a single extraction from a small portion of a tissue culture plantlet for the detection of viruses from three different families.     

Comparative detection of mild strains of potato spindle tuber viroid from the dormant potato tubers by Return-polyacrylamiee gel electrophoresis and nucleic acid hybridization

Summary Using a modified procedure of Return-polyacrylamide gel electrophoresis (R-PAGE) mild strains of potato spindle tuber viroid (M-PSTV) were detected reliably from dormant tubers. The sensitivity of R-PAGE detection of M-PSTV was equivalent to that of nucleic acid hybridization. Both methods detected M-PSTV when infected tissue was mixed with healthy tissue in a ratio of 1 to 100. When extracted nucleic acid was diluted with buffer, R-PAGE detected PSTV at a dilution of 1:256 and nucleic hybridization only up to 1:64 PSTV was readily detected from 18 potato cultivars. In addition, mild, ‘intermediate’ and severe strains were separated by R-PAGE, on the basis of their mobility on the electrophorogram.     

Detection of potato spindle tuber viroid by nucleic acid spot hybridization: Evaluation with tuber sprouts and true potato seed

A diagnostic test for the potato spindle tuber viroid (PSTV) that is based on hybridization of highly radioactive, recombinant DNA complementary to PSTV with PSTV bound to a nitrocellulose membrane and autoradiographic detection of the resulting DNA-RNA hybrids has been evaluated with tubers from 20 potato clones maintained at the International Potato Center (CIP) and with true seed obtained from healthy or PSTV-infected potato plants. The nucleic acid spot hybridization test detected PSTV not only in tuber sprouts from 10 clones that had previously tested positive in polyacrylamide gel electrophoretic (PAGE) analysis, but also in tuber sprouts from 9 clones that had previously tested negative in PAGE analysis. Further tests confirmed the presence of PSTV in these clones. The spot hybridization test detected PSTV in mixtures of seed extracts equivalent to as few as one seed from a PSTV-infected plant and 80 seeds from healthy plants. The spot hybridization test was shown to be more sensitive and reliable than PAGE analysis; it is suitable for the testing of large numbers of samples with a minimum expenditure of labor and materials.     

Detection of potato spindle tuber viroid by molecular hybridization and bioassay. A large-scale comparison

Summary To assess the reliability of dot-spot hybridization assay for the Potato Spindle Tuber Viroid, more than 1000 potato samples were cross-tested by biological and hybridization methods. Both methods gave similar results with a range of cultivars. The dot-spot hybridization test was reproducible with formaldehyde-denaturated cell-sap samples stored at room temperature for over 6 months. This finding means that samples can be transported from field stations to a diagnostic laboratory and allows for cross-checking of results between laboratories. The data suggest that dot-spot hybridization is suitable for breeding and quarantine purposes.     

Nano-mechanical transduction of polymer micro-cantilevers to detect bio-molecular interactions

Using variothermal polymer micro-injection molding, disposable arrays of eight polymer micro-cantilevers each 500 μm long, 100 μm wide and 25 μm thick were fabricated. The present study took advantage of an easy flow grade polypropylene. After gold coating for optical read-out and asymmetrical sensitization, the arrays were introduced into the Cantisens(?) Research system to perform mechanical and functional testing. We demonstrate that polypropylene cantilevers can be used as biosensors for medical purposes in the same manner as the established silicon ones to detect single-stranded DNA sequences and metal ions in real-time. A differential signal of 7 nm was detected for the hybridization of 1 μM complementary DNA sequences. For 100 nM copper ions the differential signal was found to be (36 ± 5) nm. Nano-mechanical sensing of medically relevant, nanometer-size species is essential for fast and efficient diagnosis.     

Detection of potato viruses A,M, S,X, Yand leafroll and potato spindle tuber viroid from tissue culture plantlets using single leaf discs

Using enzyme-linked immunosorbent assay (ELISA) and dotimmunobinding assay (DIBA) for potato viruses A (PVA), M (PVM), S (PVS), X (PVX), Y^N (PVY^N), Y^O (PVY^O) and leafroll (PLRV) and nucleic acid spot hybridization (NASH) for potato spindle tuber viroid (PSTVd), virus and viroid were detected reliably from single leaf discs (6 mm) of tissue-culture plantlets. Leaf discs taken from leaf positions (1 to 8) (bottom to top) can be used for detection of all viruses except PLRV where the lower leaves had higher concentrations of virus than the leaves from the upper part of the plantlet. Virus cultures were maintained for 1 to 4 years in several potato cultivars. The levels of virus remained reproducible except for PVM concentration, which was found to be very low in cv. Green Mountain. Using densitometry software, the DIBA spots were quantified and results were comparable to A₄₀₅ values obtained by ELISA. PSTVd concentration as measured by densitometry from spots of NASH indicated no loss of viroid over 1–4 years in tissue culture in two potato cultivars.     

Chemiluminescent detection of potato spindle tuber viroid in true potato seed using a digoxigenin labelled DNA probe

Summary The potential use of a simple, sensitive and non-radioactive method for detecting potato spindle tuber viroid (PSTVd) in germinated true potato seeds, based on nucleic acid hybridization with a PSTVd-specific DNA probe labelled with digoxigenin, was investigated. Two simple procedures for the clarification of plant extracts suitable for a non-radioactive detection system were also investigated. The nucleic acid hybridization technique could detect one PSTVd-infected seed in more than 150 healthy seeds. The benefits of this non-radioactive detection system are discussed.     

Extreme resistance to infection by potato virus X in genotypes of wild tuber-bearingSolanum species

Summary Clones derived from thirty-one different accessions (nineteen of Argentine origin) belonging to eightSolanum species were screened for resistance to infection by potato virus X strain cp (PVX cp) by mechanical inoculation of plantlets that had been micropropagated in vitro. Estimates of PVX multiplication obtained by enzyme linked immunosorbent assay and slot blot nucleic acid hybridization allowed the identification of resistant clones derived from five accessions belonging toS. commersonii S. oplocense, S. sparsipilum andS. tuberosum andigena. Resistant genotypes supported PVX concentrations 5 to 15 times smaller than did the susceptible control cultivar Spunta. Graft inoculation test confirmed the presence of extreme resistance similar to that conferred by the ‘immunity’ gene X¹ (also called RX_act).     

Comparison of Pollen Grain Treatments Without Mechanical Fracturation Prior to Protein Quantification

Protein and amino acids in pollen are important nutritional components for larval development in several insect species, especially in Apoidea. The Bradford assay is a widely used method to measure relative protein content of pollen, which can shed light on pollen quality and consequences to fitness. Prior to using the Bradford assay, protein must be released from pollen grains, often using a mixture of chemical and mechanical fracturation methods. In this study, we tested the efficacy of protein extraction without using mechanical fracturation. We used pollen collected by the solitary bee Osmia lignaria Say to compare two known buffers associated with pollen protein analysis: phosphate-buffered saline and sodium hydroxide, and deionized water, and with different pollen weights from which we quantified protein using the Bradford assay. While all buffers and deionized water were useful in releasing protein from pollen grains collected by O. lignaria, the use of sodium hydroxide resulted in significantly higher protein quantification across all pollen weights. This methodological study can inform future studies of pollen nutrition in pollen-foraging species.     

基于亲水相互作用液相色谱-三重四极杆质谱法研究白茶萎凋过程中代谢物的变化

采用亲水相互作用液相色谱-三重四极杆质谱法,对白茶鲜叶(F)和室内萎凋处理29 h(W)、53 h(P)的2个萎凋样进行了代谢物靶向检测,Quality control(QC)样本中共检测到111个代谢物,主要包括47种氨基酸及其衍生物、29种核酸类代谢物、12种维生素和辅酶、17种糖代谢物和6种甘油磷脂代谢物。与鲜叶F相比,萎凋叶W和P中显著增加的代谢物分别有39种和41种,其中共有代谢物35种;显著降低的代谢物分别有9种和11种,其中共有代谢物8种。显著变化代谢物分析结果表明,萎凋叶的细胞膜受损、DNA和RNA降解、氨基酸衍生物显著增加、糖代谢异常。根据代谢物含量的变化幅度和组成特异性,筛选了表征萎凋叶细胞膜生理状态和核酸、糖代谢的标志代谢物。此外,针对代谢物中氨基酸和核苷酸的组成特点,提出了研究氨基酸衍生物滋味特性的必要性和应用代谢谱分析建立萎凋工艺适度标准的可能性。     

Efficient Purification of R-phycoerythrin from Marine Algae (Porphyra yezoensis) Based on a Deep Eutectic Solvents Aqueous Two-Phase System

R-phycoerythrin (R-PE), a marine bioactive protein, is abundant in Porphyra yezoensis with high protein content. In this study, R-PE was purified using a deep eutectic solvents aqueous two-phase system (DES-ATPS), combined with ammonium sulphate precipitation, and characterized by certain techniques. Firstly, choline chloride-urea (ChCl-U) was selected as the suitable DES to form ATPS for R-PE extraction. Then, single-factor experiments were conducted: the purity (A₅₆₅/A₂₈₀) of R-PE was 3.825, and the yield was 69.99% (w/w) under optimal conditions (adding 0.040 mg R-PE to ChCl-U (0.35 g)/K₂HPO₄ (0.8 g/mL, 0.5 mL) and extracting for 20 min). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that the purified R-PE contained three main bands. One band was presented after purification in native-PAGE. The UV-vis spectra showed characteristic absorption peaks at 495, 540, and 565 nm. R-PE displayed an emission wavelength at 570 nm when excited at 495 nm. All spectra results illustrated that the structure of R-PE remained unchanged throughout the process, proving the effectiveness of this method. Transmission electron microscope (TEM) showed that aggregation and surrounding phenomena were the driving forces for R-PE extraction. This study could provide a green and simple purification method of R-PE in drug development.     

The formation and UV-blocking property of flower-like ZnO nanorod on electrospun natural cotton cellulose nanofibers

We report a simple and effective route to fabricate branched hierarchical flower-like nanostructures of ZnO on natural cotton cellulose fiber by combining electrospinning and the low-temperature hydrothermal growth technique. First, natural cotton cellulose nanofibers were prepared by electrospinning cotton cellulose /LiCl/DMAc solution. The electrospun cotton cellulose nanofibers served as flexible substrate, on which the branched, highly uniform, and dense flower-like ZnO were hydrothermally grown. The as-prepared cotton cellulose/ZnO nanocomposite fibers were characterized by SEM, HRTEM, EDS, TG, and UV-vis spectrophotometry. The modified cotton cellulose nanocomposite fibers were not only exhibiting dispersed uniformly, but also rendered excellent protection against UV radiation because of the incorporation of flower-like ZnO nanostructures. Therefore, the as-prepared nanocomposite fibers demonstrate a significant performance in ultraviolet protection and provide a potential application for ultraviolet detection.     

The effect of the inhibitors of protein and nucleic acid synthesis on the coumarin-induced tuberization and growth of excised axillary shoots of potato sprquts (Solanum tuberosum l.) culturedin vitro

The inhibitors of protein and nucleic acid synthesis; chloramphenicol, para-fluorophenylalanine, actinomycin D, and 5-fluorouracil were evaluated for their effectiveness in inhibiting coumarin-induced tuberization, tuber growth, and the inhibition of.root and shoot growth of excised axillary shoots of potato sprouts culturedin vitro. To date, it has been demonstrated that both kinetin and coumarin readily effect 100 percent tuberization of the shoots culturedin vitro. The results of the present study demonstrate that with the exception of 5-fluorouracil, the inhibitors significantly inhibited the coumarin-induced. tuberization, tuber growth, and root growth whereas in previously published data by Palmer and Smith (6), the inhibitors merely delayed kinetin-induced tuberization. It is suggested that both protein and nucleic acid synthesis are required for the coumarin-induced tuberization processes, and the mode of action of coumarin is distinctly different from that of kinetin.     

水稻愈伤组织生长和衰老过程中信息大分子化合物的变化

 水稻愈伤组织转移继代培养后的0—20天,生长速率较快,核酸和蛋白质含量急剧增加,且维持较高水平,核糖核酸酶和血红蛋白水解酶活性急剧下降,且维持较低活性。因此,愈伤组织的继代培养时间最好掌握在生长速率较快,核酸蛋白质含量较高、水解酶类活性较低的阶段,即在20天以内。核酸和蛋白质含量与其水解酶类(核糖核酸酶和血红蛋白水解酶)的比活之间存在着密切的关系,从而认为核糖核酸酶和血红蛋白水解酶分别在核酸和蛋白质含量下降中起着积极作用。根据信息大分子化合物的变化动态和愈伤组织外部形态变化提出愈伤组织转移继代培养过程可以分为三个阶段,即旺盛生长、稳定生长和衰老阶段。     

Selective cationic surfactant detection in aqueous solution by polyurethane copolymer linked with metal ion indicator

Polyurethane (PU) copolymer is laterally linked with three kinds of metal ion indicator (calcein, calmagite, or eriochrome black T), with which free metal ion in aqueous solution is intended to be detected by PU color change. Metal ion detection by the indicator-PU fails due to the poor permeation of hydrophilic metal ion into hydrophobic PU layer. Instead, three surfactants with different ionic head groups, aerosol OT (AOT), cetyltrimethylammonium bromide (CTAB), and sodium dodecylsulfate (SDS), are tested for metal ion. Cationic CTAB exhibits an instant PU color change, but anionic AOT and SDS do not respond at all. Reason for the selective detection of cationic surfactant is the complex formation between cationic surfactant and indicator. Molecular interactions between PUs are affected by the laterally linked indicators based on the results by infrared spectra and differential thermal analysis. UV-vis spectra reveal that extra peak arising from the linked indicator appears compared to plain PU. The lateral linking of indicator to PU demonstrates, as well as the selective surfactant detection, a 454 % increase in tensile strength and reproducible shape recovery as high as 99 % compared to plain PU.