Trifolium pratense
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Commonly studied in the context of legume-rhizobia symbiosis, biological nitrogen fixation (BNF) is a key component of the nitrogen cycle in nature. Despite its potential in plant breeding and many years of research, information is still lacking as to the regulation of hundreds of genes connected with plant-bacteria interaction, nodulation, and nitrogen fixation. Here, we compared root nodule transcriptomes of red clover (Trifolium pratense L.) genotypes with contrasting nitrogen fixation efficiency, and we found 491 differentially expressed genes (DEGs) between plants with high and low BNF efficiency. The annotation of genes expressed in nodules revealed more than 800 genes not yet experimentally confirmed. Among genes mediating nodule development, four nod-ule-specific cysteine-rich (NCR) peptides were confirmed in the nodule transcriptome. Gene duplication analyses revealed that genes originating from tandem and dispersed duplication are significantly over-represented among DEGs. Weighted correlation network analysis (WGCNA) organized expression profiles of the transcripts into 16 modules linked to the analyzed traits, such as nitrogen fixation efficiency or sample-specific modules. Overall, the results obtained broaden our knowledge about transcriptomic landscapes of red clover's root nodules and shift the phenotypic description of BNF efficiency on the level of gene expression in situ.
- Publikační typ
- časopisecké články MeSH
Plant-rhizobia symbiosis can activate key genes involved in regulating nodulation associated with biological nitrogen fixation (BNF). Although the general molecular basis of the BNF process is frequently studied, little is known about its intraspecific variability and the characteristics of its allelic variants. This study's main goals were to describe phenotypic and genotypic variation in the context of nitrogen fixation in red clover (Trifolium pretense L.) and identify variants in BNF candidate genes associated with BNF efficiency. Acetylene reduction assay validation was the criterion for selecting individual plants with particular BNF rates. Sequences in 86 key candidate genes were obtained by hybridization-based sequence capture target enrichment of plants with alternative phenotypes for nitrogen fixation. Two genes associated with BNF were identified: ethylene response factor required for nodule differentiation (EFD) and molybdate transporter 1 (MOT1). In addition, whole-genome population genotyping by double-digest restriction-site-associated sequencing (ddRADseq) was performed, and BNF was evaluated by the natural 15N abundance method. Polymorphisms associated with BNF and reflecting phenotype variability were identified. The genetic structure of plant accessions was not linked to BNF rate of measured plants. Knowledge of the genetic variation within BNF candidate genes and the characteristics of genetic variants will be beneficial in molecular diagnostics and breeding of red clover.
- MeSH
- alely MeSH
- fenotyp MeSH
- fixace dusíku genetika MeSH
- genotyp MeSH
- interakce mikroorganismu a hostitele MeSH
- kořeny rostlin genetika mikrobiologie MeSH
- polymorfismus genetický * MeSH
- Rhizobium fyziologie MeSH
- rostlinné geny genetika MeSH
- sekvenční analýza DNA metody MeSH
- symbióza genetika MeSH
- Trifolium genetika mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
Východiska: Ferritin je globulární vnitrobuněčný protein, který slouží jako hlavní zásobárna železa. U nádorových onemocnění je plazmatická koncentrace ferritinu zvýšená. V řadě studií bylo zjištěno, že nádorové buňky exprimují ve zvýšené míře transferinové receptory (TfR). Zvýšená exprese TfR byla pozorována i u karcinomu prostaty. Apoferritin (APO) lze využít jako proteinový nanotransportér, do kterého je možné enkapsulovat vhodnou léčivou látku. Bylo zjištěno, že nanočástice zvyšují prostupnost nanotransportérů do nádorové buňky a vykazují fototermální efekt. Cílem projektu bylo enkapsulovat doxorubicin (DOX) do APO a vytvořený APO/DOX modifikovat zlatými (gold nanoparticles - AuNPs) a stříbrnými (silver nanoparticles prepared by green synthesis - AgNPsGS) nanočásticemi. Metody: Pro charakterizaci APO byla použita 10% gelová elektroforéza (sodium dodecylsulphate polyacrylamide gel electrophoresis - SDS-PAGE) - 120 V, 60 min, 24 mM Tris, 0,2 M glycin, 3 mM SDS. Fluorescence DOX (Ex 480 nm, Em 650 nm) s typickým absorpčním maximem v 560 nm. Elektrochemické měření bylo provedeno v Brdičkově roztoku (tříelektrodové zapojení). AgNPsGS byly připraveny zelenou syntézou z jetele lučního (Trifolium pratense L.). Výsledky: Byla provedena elektroforetická studie APO a APO/DOX (5- 100 μg//ml). Bylo sledováno chování APO a APO/DOX (10 μM) v závislosti na pH. APO v kyselém prostředí tvoří subjednotky o velikosti asi 20 kDa a v neutrálním a zásaditém prostředí se zformuje na globulární protein o velikosti asi 450 kDa. U APO/DOX byla pozorována změna mobility (asi o 10 %). Na povrchu APO/DOX byl vytvořen film z AuNPs. APO/DOX/AuNPs byl následně promyt ultračistou vodou. Bylo sledováno uvolnění DOX v závislosti na pH. Množství analyzovaného DOX se zvýšilo až o 50 %. Dále byl navržen a připraven komplex AgNPsGS-DOX (1 mg AgNPsGS/100 μM DOX). Takto připravený komplex AgNPsGS-DOX byl uzavřen do APO. Pro další zlepšení terapeutické účinnosti byl připravený komplex APO/AgNPsGS-DOX pokryt vrstvou AuNPs. Vytvořený APO/AgNPsGS-DOX/AuNPs prokázal svoji stabilitu a při změně fyzikálních parametrů byl z komplexu uvolněn DOX. Závěr: Byly připraveny a modifikovány nanokomplexy APO pro zvýšení terapeutické účinnosti protinádorové léčby. Zacílení k nádorové buňce bylo na TfR nebo efektem zvýšené propustnosti a retence. Uvolnění léčiva bylo možné změnou pH nebo fototermální aktivací, která bude nyní testována.
Background: Ferritin is a globular intracellular protein that acts as the main reservoir for iron. Malignancies are associated with increased plasma ferritin concentrations. A number of studies show that tumor cells express high levels of transferrin receptors (TfR). Increased TfR expression was observed in prostate carcinoma. Apoferritin (APO) can be used as a protein nanotransporter into which a suitable medicinal substance can be encapsulated. Nanoparticles increase the permeability of tumor cells to nanotransporters and have a photothermal effect. The aim of this study was to encapsulate doxorubicin (DOX) into APO and to modify the resulting APO/DOX with gold (AuNPs) and silver nanoparticles prepared by green synthesis (AgNPsGS). Methods: APO was characterized using 10% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS- -PAGE) – 120 V, 60 min, 24 mM Tris, 0.2 M glycine, 3 mM SDS. DOX fluorescence (Ex 480 nm; Em 650 nm) was observed, with a typical absorption maximum at 560 nm. Electrochemical measurement was performed in Brdicka solution (three-electrode setup). AgNPsGS were prepared by green synthesis using clover (Trifolium pratense L.). Results: An electrophoretic study of APO and APO/DOX (5–100 μg/mL) was performed and the behavior of APO and APO/DOX (10 μM) as a function of pH was monitored. In an acidic environment, APO forms subunits of about 20 kDa; in an alkaline medium, it forms a globular protein of about 450 kDa. A change in APO/DOX mobility (about by 10%) was observed. A fi lm of gold nanoparticles was applied to the APO/DOX surface. APO/DOX-AuNPs were washed with ultra-pure water. pH-dependent release of DOX a was monitored. The amount of DOX analyzed was increased by up to 50%. Furthermore, an AgNPsGS-DOX complex (1 mg AgNPsGS/100 μM DOX) was generated and prepared. Subsequently, the AgNPsGS-DOX complex was encapsulated into APO. To further improve therapeutic efficacy, the APO/AgNPsGS-DOX complex was coated with an Au layer. APO/AgNPsGS-DOX/AuNPs were stable and DOX was released from the complex after physical parameters had changed. Conclusion: APO nanocomplexes were prepared and modified to increase therapeutic efficacy against tumors. Tumor cell targeting was achieved by binding to TfR and via increased tumor cell permeability and retention. Release of the drug was made possible due to a pH change and photothermal activation that will now be tested.
- Klíčová slova
- nanotransportér, poferritinový nanotransportér, transferinové receptory, stříbrné nanočástice, zlaté nanočástice,
- MeSH
- apoferritiny farmakologie MeSH
- kovové nanočástice MeSH
- lékové transportní systémy * metody MeSH
- lidé MeSH
- nádory prostaty * farmakoterapie MeSH
- nanomedicína * metody MeSH
- objevování léků MeSH
- stříbro MeSH
- zlato MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
KEY MESSAGE: Vanadium compounds increased the content and release of distinct isoflavones in a Trifolium pratense suspension culture. Regarding transport-mechanism inhibitors, the process was mostly facilitated by ABC proteins and vesicular transport. The transport of isoflavones and other secondary metabolites is an important part of metabolism within plants and cultures in vitro regarding their role in defence against various abiotic and biotic stressors. This research focuses on the way how to increase production and exudation of isoflavones by application of chemical elicitor and the basic identification of their transport mechanisms across cell membranes. The release of five isoflavones (genistin, genistein, biochanin A, daidzein, and formononetin) into a nutrient medium was determined in a Trifolium pratense var. DO-8 suspension culture after two vanadium compound treatments and cultivation for 24 and 48 h. The NH4VO3 solution caused a higher concentration of isoflavones in the medium after 24 h. This increased content of secondary metabolites was subsequently suppressed by distinct transport-mechanism inhibitors. The transport of isoflavones in T. pratense was mostly affected by ABC inhibitors from the multidrug-resistance-associated protein subfamily, but the genistein concentration in the medium was lower after treatment with multidrug-resistance protein subfamily inhibitors. Brefeldin A, which blocks vesicular transport, also decreased the concentration of some isoflavones in the nutrient medium.
Using high-throughput sequencing, a novel waikavirus was identified in a mixed virus infection of red clover (Trifolium pratense L.). Its complete genomic sequence was determined and characterized. The virus, tentatively named red clover associated virus 1 (RCaV1), is phylogenetically related to members of the genus Waikavirus (family Secoviridae, order Picornavirales).
- MeSH
- fylogeneze MeSH
- genom virový * MeSH
- molekulární sekvence - údaje MeSH
- nemoci rostlin virologie MeSH
- otevřené čtecí rámce MeSH
- satelitní viry klasifikace genetika izolace a purifikace MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- Trifolium virologie MeSH
- Waikavirus klasifikace genetika izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
This study reports the complete genomic sequence of a novel virus isolated from red clover. According to its genomic organization, its similarity to luteoviruses, and a greater than 10% difference in all genes, this virus isolate likely represents a new luteovirus species. As seen in nectarine stem pitting-associated virus (NSPaV) and NSPaV-South Korea (SK) luteoviruses, it differs from typical luteoviruses through the absence of ORF3a and ORF4 encoding movement proteins. Furthermore, its P5 protein (responsible for aphid transmission) is more similar to the P5 of enamoviruses than that of luteoviruses. The virus isolate has been named red clover-associated luteovirus (RCaV).
During aetiological study of diseased red clover (Trifolium pratense L.) using high throughput sequencing, a novel virus with a 10 kb genome divided into two segments was discovered. The virus, tentatively named red clover associated varicosavirus (RCaVV), is phylogenetically related to classifiable members of the genus Varicosavirus (family Rhabdoviridae, order Mononegavirales). Analysis of mRNA levels from the individual RCaVV genes suggested possible differences in transcription regulation between rhabdoviruses with divided and undivided genomes.
- MeSH
- DNA viry klasifikace genetika izolace a purifikace MeSH
- fylogeneze MeSH
- genom virový MeSH
- nemoci rostlin virologie MeSH
- rostlinné viry klasifikace genetika izolace a purifikace MeSH
- sekvenční analýza DNA MeSH
- Trifolium virologie MeSH
- virové proteiny genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
During high throughput sequencing (HTS) of leaves from a symptomatic red clover plant, a new RNA virus, tentatively named red clover nepovirus A (RCNVA), was discovered. The complete genomic sequence was determined and characterized. Particularly noteworthy was that RCNVA shares high sequence identities in RNA1 with a group of phylogenetically related nepoviruses while homologies in the RNA2 segments are markedly lower. Based on the genomic organization and phylogenetic attributes, RCNVA should be classified as a novel virus of the genus Nepovirus (subfamily Comovirinae, family Secoviridae, order Picornavirales).
In the production of fermented feed, each crop can be contaminated with a variety of microorganisms that may produce natural pollutants. Biogenic amines, mycotoxins, and undesirable organic acids can decrease health feed safety. The aim of this study was to compare the counts of microorganisms, levels of biogenic amines, and the mycotoxins in forage legumes, and also to compare the occurrence of microorganisms and levels of mycotoxins in green fodder and subsequently produced silage and the influence of additives on the content of natural harmful substances in silage. The experimental plot was located in Troubsko and Vatín, in the Czech Republic. Two varieties of Medicago sativa and one variety of Trifolium pratense were compared. Green fodder and subsequently produced silage reaching up to 23% of dry matter were evaluated and prepared using a bio-enzymatic additive and a chemical additive. Green fodder of Medicago sativa was more contaminated by Enterococci than Trifolium pratense fodder. The obvious difference was determined by the quality of silage leachate. The silage prepared from Medicago sativa fodder was more contaminated with butyric acid. Fungi were present in higher counts in the anaerobic environment of green fodder and contaminated it with zearalenone and deoxynivalenol. Lower counts of fungi were found in silage, although the zearalenone content did not change. Lower content of deoxynivalenol was detected in silage, compared with green fodder. Silages treated with a chemical additive were found not to contain butyric acid. Lower ethanol content was determined, and the tendency to reduce the risk of biogenic amines occurrence was evident. The additives proved to have no influence on the content of mycotoxins.
- MeSH
- biogenní aminy chemie MeSH
- fermentace MeSH
- kontaminace potravin prevence a kontrola MeSH
- krmivo pro zvířata mikrobiologie MeSH
- Medicago sativa chemie mikrobiologie MeSH
- mykotoxiny chemie MeSH
- potravinářské přísady farmakologie MeSH
- siláž analýza mikrobiologie MeSH
- Trifolium chemie mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Subterranean clover is an important annual forage legume, whose diploidy and inbreeding nature make it an ideal model for genomic analysis in Trifolium. We reported a draft genome assembly of the subterranean clover TSUd_r1.1. Here we evaluate genome mapping on nanochannel arrays and generation of a transcriptome atlas across tissues to advance the assembly and gene annotation. Using a BioNano-based assembly spanning 512 Mb (93% genome coverage), we validated the draft assembly, anchored unplaced contigs and resolved misassemblies. Multiple contigs (264) from the draft assembly coalesced into 97 super-scaffolds (43% of genome). Sequences longer than >1 Mb increased from 40 to 189 Mb giving 1.4-fold increase in N50 with total genome in pseudomolecules improved from 73 to 80%. The advanced assembly was re-annotated using transcriptome atlas data to contain 31 272 protein-coding genes capturing >96% of the gene content. Functional characterization and GO enrichment confirmed gene expression for response to water deprivation, flavonoid biosynthesis and embryo development ending in seed dormancy, reflecting adaptation to the harsh Mediterranean environment. Comparative analyses across Papilionoideae identified 24 893 Trifolium-specific and 6325 subterranean-clover-specific genes that could be mined further for traits such as geocarpy and grazing tolerance. Eight key traits, including persistence, improved livestock health by isoflavonoid production in addition to important agro-morphological traits, were fine-mapped on the high-density SNP linkage map anchored to the assembly. This new genomic information is crucial to identify loci governing traits allowing marker-assisted breeding, comparative mapping and identification of tissue-specific gene promoters for biotechnological improvement of forage legumes.
- MeSH
- genom rostlinný genetika MeSH
- genomika metody MeSH
- sekvenční analýza DNA metody MeSH
- Trifolium genetika MeSH
- Publikační typ
- časopisecké články MeSH
