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Purification and properties of alpha-D-mannosidase from the germinated seeds of Medicago sativa (alfalfa).

ABSTRACT: alpha-Mannosidase of Medicago sativa (alfalfa) was purified 1340-fold. The purification method included dialysis of the crude extract against a citrate/phosphate buffer, pH 3.9, (NH4)SO4 precipitation, hydroxyapatite chromatography, chromatography on Sephadex G-200 and finally a preparatory electrophoresis on polyacrylamide-gel gradient by Doly & Petek's [(1977) J. Chromatogr. 137. 69--81] method. Each step of purification was checked by polyacrylamide-gel disc electrophoresis. The purified enzyme showed a single band, corresponding to alpha-mannosidase activity. alpha-Mannosidase has a mol.wt. 230 000 as estimated by Hedrick & Smith's [(1968) Arch. Biochem. Biophys. 126, 155--164] method and also by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by Weber & Osborn [(1969) J. Biol. Chem. 244, 4406--4412]. The enzyme comprises four subunits of different molecular weight. Optimum pH and Km values were determined with p-nitrophenyl alpha-D-mannoside as substrate. When incubated at a temperature between 20 and 62 degrees C before assay, alpha-mannosidase initially shows an increase in activity. alpha-Mannosidase is stable when the pH is about neutrality. It can be inactivated by several metal ions, including Zn2+. At a pH below 5 the enzyme undergoes irreversible inactivation. The presence of EDTA at acid pH considerably enhances the inactivation of the enzyme. This inactivation due to EDTA can be specifically reversed by incubation with Zn2+.

SUBMITTER: Curdel A

PROVIDER: S-EPMC1161373 | biostudies-other | 1980 Feb

REPOSITORIES: biostudies-other

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Project description:BackgroundNucleorhabdoviruses possess bacilliform particles which contain a single-stranded negative-sense RNA genome. They replicate and mature in the nucleus of infected cells. Together with viruses of three other genera of the family Rhabdoviridae, they are known to infect plants and can be transmitted by arthropod vectors, during vegetative propagation, or by mechanical means. In 2010, an alfalfa (Medicago sativa) plant showing virus-like symptoms was collected from Stadl-Paura, Austria and sent to Julius Kühn Institute for analysis.MethodsElectron microscopy (EM) of leaf extracts from infected plants revealed the presence of rhabdovirus-like particles and was further used for ultrastructural analyses of infected plant tissue. Partially-purified preparations of rhabdovirus nucleocapsids were used for raising an antiserum. To determine the virus genome sequence, high throughput sequencing (HTS) was performed. RT-PCR primers were designed to confirm virus infection and to be used as a diagnostic tool.ResultsEM revealed bacilliform virions resembling those of plant-infecting rhabdoviruses. HTS of ribosomal RNA-depleted total RNA extracts revealed a consensus sequence consisting of 13,875 nucleotides (nt) and containing seven open reading frames (ORFs). Homology and phylogenetic analyses suggest that this virus isolate represents a new species of the genus Nucleorhabdovirus (family Rhabdoviridae). Since the virus originated from an alfalfa plant in Austria, the name alfalfa-associated nucleorhabdovirus (AaNV) is proposed. Viroplasms (Vp) and budding virions were observed in the nuclei of infected cells by EM, thus confirming its taxonomic assignment based on sequence data.ConclusionsIn this study, we identified and characterised a new nucleorhabdovirus from alfalfa. It shared only 39.8% nucleotide sequence identity with its closest known relative, black currant-associated rhabdovirus 1. The virus contains an additional open reading frame (accessory gene) with unknown function, located between the matrix protein and the glycoprotein genes. Serological and molecular diagnostic assays were designed for future screening of field samples. Further studies are needed to identify other natural hosts and potential vectors.

Project description:Agronomic and quality traits in alfalfa are very important to forage industry. Genomic prediction (GP) based on genotyping-by-sequencing (GBS) data could shorten the breeding cycles and accelerate the genetic gains of these complex traits, if they display moderate to high prediction accuracies. The aim of this study was to investigate the predictive potentials of these traits in alfalfa. A total of 322 genotypes from 75 alfalfa accessions were used for GP of the agronomic and quality traits, which were related to yield and nutrition value, respectively, using BayesA, BayesB, and BayesC? methods. Ten-fold cross validation was used to evaluate the accuracy of GP represented by the correlation between genomic estimated breeding value (GEBV) and estimated breeding value (EBV). The accuracies ranged from 0.0021 to 0.6485 for different traits. For each trait, three GP methods displayed similar prediction accuracies. Among 15 quality traits, mineral element Ca had a moderate and the highest prediction accuracy (0.34). NDF digestibility after 48 h (NDFD 48 h) and 30 h (NDFD 30 h) and mineral element Mg had prediction accuracies varying from 0.20 to 0.25. Other traits, for example, fat and crude protein, showed low prediction accuracies (0.05 to 0.19). Among 10 agronomic traits, however, some displayed relatively high prediction accuracies. Plant height (PH) in fall (FH) had the highest prediction accuracy (0.65), followed by flowering date (FD) and plant regrowth (PR) with accuracies at 0.52 and 0.51, respectively. Leaf to stem ratio (LS), plant branch (PB), and biomass yield (BY) reached to moderate prediction accuracies ranging from 0.25 to 0.32. Our results revealed that a few agronomic traits, such as FH, FD, and PR, had relatively high prediction accuracies, therefore it is feasible to apply genomic selection (GS) for these traits in alfalfa breeding programs. Because of the limitations of population size and density of SNP markers, several traits displayed low accuracies which could be improved by a bigger reference population, higher density of SNP markers, and more powerful statistic tools.

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Purification and properties of alpha-D-mannosidase from the germinated seeds of Medicago sativa (alfalfa).

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