1,720,974 research outputs found
Development of method of RNA extraction from leaves for gene expression analysis of domestic apple
No full textRealaus laiko PGR (RL-PGR) yra metodas taikomas genų raiškos tyrimui – nustatyti vyksta ar nevyksta tiriamų geno raiška, geno raiškos kiekybiniam įvertinimui įvairių veiksnių poveikyje, kaip skiriasi įvairių genų raiška tarpusavyje. Norint ištirti augalų genų raišką reikia išgryninti augalo RNR, paruošti kDNR ir kiekybiškai įvertinti tiriamo geno raiškos lygi taikant RL-PGR analizės metodą. Naminė obelis (lot. Malus x domestica Borkh.) – erškėtinių šeimos augalas, vienas ekonomiškai svarbiausių sodininkystėje naudojamų augalų rūšių. Malus sp. augalams būdingas kiekybinis ir kokybinis atsparumas patogenams, todėl genų, sąlygojančių atsparumą patogenams, identifikavimas, tyrimas ir perkėlimas iš laukinių obels rūšių į naminės obels veisles yra pagrindinis obelų selekcijos programų akcentas. Šiuo darbu buvo pritaikytas RNR gryninimo metodas tinkamas RNR ir kDNR paruošimui iš obels lapų audinių ir optimizuotos RL-PGR analizės sąlygos obels pavyzdžiams. Apibendrinti mokslinėje literatūroje paskelbti RNR paruošimo metodai ir parinktos tinkamos salygos RNR gryninimui iš naminės obels lapų. RNR kokybė įvetinta spektrafotometriškai ir pagal 18S ir 25S rRNR stabilumą. Tyrimas parodė, kad didžiausia RNR išeiga ir geriausią kokybė gauta taikant sąlygas naudotas Yu ir kt. (2012) ir Louime ir kt. (2008) paskelbtuose metoduose: esktrakcija vykdyta pH 9,0 tirpalu su 1,4 M NaCl ir 3% CTAB, tuomet RNR ekstrahuota chloroformu. Tolimesnis RNR išsodinimas LiCl2 tirpalu užtikrina geriausią RNR kokybę ir išeiga kai taikoma 3 val. inkubacija -20 oC temperatūroje. Saugojant -70 oC temperatūroje RNR pavyzdžiai išlieka stabilūs vandeniniame tirpale ir pavyzdžio išeiga yra didesnė nei išsodinus 95% etanoliu. Įvertintas nuolat aktyvių genų („housekeeping genai“, HKG) taikymas naminės obels pavyzdžių RL-PGR analizei. Nustatyta, kad naminės obels pavyzdžių RL-PGR duomenų normalizavimui yra tinkamas elongacijos faktoriaus - 1 (EF - 1) genas.RL-PCR is a method for detection of gene expression used to identify if expression of investigated gene occurs or not, for quantitative evaluation of gene expression to assess how multiple factors change gene expression and how differs expression of various gene among each other. To investigate expression of plant gene, it is required to purify plant RNA, prepare cDNA and quantitative evaluation of level of gene expression is performed using RT-PCR analysis method. Domestic apple (Malus x domestica Borkh.) – is a plant of Rosaceae family that is one of the most important species used in horticulture. Quantitative and qualitative resistance to scab traits are characteristic to plants of Malus sp., therefore transfer of the genes responsible for resistance from wild species to apple cultivars is the main focus of apple breeding programs. The goal of this work was to adapt RNA purification method, suitable for RNA and cDNA preparation from apple leaf tissue and to optimize RT-PCR analysis conditions for apple samples. RNA preparation methods, published in scientific literature were summarized and appropriate conditions for the purification of RNA from apple leaves were selected. RNA quality was assessed by evaluating stability of 18S and 25S rRNA. The study showed that the highest RNA yield and the best quality was obtained using conditions described by Yu et al. (2012) and Louime and et al. (2008): extraction was performed using pH 9.0 solution with 1.4 M NaCl and 3% CTAB, and chloroform was used for RNA extraction. Further RNA precipitation with LiCl2 solution ensures the best RNA quality and yield when applied as 3 hour incubation at –20 °C temperature. RNA sample storage at –70 °C temperature ensures stability in aqueous solution and the yield obtained is higher as compared to precipitation with 95% ethanol. The application of constant expression genes (housekeeping gene, HKG) for RT-PCR analysis of apple leave samples was assessed. It was etermined that the EF - 1 gene is suitable for normalization of RT-PCR data of apple leave samples
Cultivated tobacco (Nicotiana tabacum) endophytic bacteria isolation and identification
No full textEndofitiniai mikroorganizmai su augalu sudaro simbiotinį ryšį ir gyvena jame didžiąją gyvenimo ciklo dalį, nesukeldami jokio žalingo poveikio. Endofitinės bakterijos yra svarbios augalų mikroekosistemų komponentai, suteikia teigiamą poveikį augalų augimui ir vystymuisi. Veikdamos tiesiogiai, pagerina maistinių medžiagų įsisavinimą, moduliuoja su augimu ar stresu susijusių hormonų išskyrimą. Netiesiogiai veikdamos, reguliuoja patogenų kiekį ir sukelia augalo gynybines reakcijas prieš juos. Šių natūralių simbiontų naudojimas suteikia galimybę padidinti augalų pasėlių produktyvumą ir sumažinti žemės ūkio poveikį aplinkai.
Šio tyrimo tikslas yra išskirti ir identifikuoti tabako (Nicotiana tabacum) augalo endofitines bakterijas. Iš viso išskirta 49 bakterijų izoliatai ir jie identifikuoti, remiantis 16S rRNR geno sekoskaitos metodu. Nustatyta, jog labiausiai dominavo Bacillus genčiai priklausančios bakterijos. Buvo identifikuota 10 rūšių: B. pumilus, B. anthracis, B. cereus, B. toyonesis, B. thuringiensis, B. mycoides, B. marisflavi, B. aquimaris, B. simplex, B. muralis.Endophytes are microbial symbionts residing within the plant for the majority of their life cycle without causing any harmful effects to the host plant. Endophytic bacteria are the important components of plant micro–ecosystems. They can be benefitial to plants directly by improving plant nutrient uptake, modulating growth and stress related phytohormones. Indirectly, they can improve plant health by targeting pathogens and by priming plant defenses. The use of these natural symbionts gives an opportunity to maximize crop productivity while reducing the environmental impacts of agriculture.
The aim of this study is to isolate and identify endophytic bacteria from tobacco plant. A total of 49 isolates were isolated from tobacco (Nicotiana tabacum) and identified based on 16S rRNA gene sequences. Bacillus sp. was dominant bacteria in tobacco. 10 species: B. pumilus, B. anthracis, B. cereus, B. toyonesis, B. thuringiensis, B. mycoides, B. marisflavi, B. aquimaris, B. simplex and B. muralis were indentified.Gamtos mokslų fakultetas / Faculty of Natural SciencesVytauto Didžiojo universitetas / Vytautas Magnus UniversityBiologijos katedra / Department of Biolog
Genes of NADPH-oxidase and Their Expression in Domestic Apple (Malus × domestica Borkh.)
No full textGenai, koduojantys NADPH-oksidazę (NOX), buvo aptikti įvairiose augalų rūšyse, o transkripcinė šių genų reguliacija parodyta esant augalų atsakui į stresą ar fitohormonų poveikį. In vitro auginimo sąlygos dažnai augalui sukelia stresą, kuris lėtina augalo augimą ir sukelia senėjimą. Aktyvių deguonies junginių gamyba (ADJ) yra svarbus procesas augalo patiriamo streso metu. Rboh (angl. respiratory burst oxidase homolog) homologų raiška augalo audiniuose augintuose in vitro sąlygomis ir jų atsakas į abiotinį stresą nėra pilnai ištirtas. Šio tyrimo tikslas buvo identifikuoti naminės obels rboh genus (Mdrboh) ir apibūdinti jų raišką obels audiniuose bei senstant ūglių kultūrai auginamai in vitro sąlygomis. Baltymų sekų panašumų paieškos metu, panaudojus baltažiedžio vairenio (Arabidopsis thaliana) rboh (Atrboh) baltymų sekas, identifikuoti devyni obels rboh genai. Filogenetine analize keturių erškėtinių šeimos augalų ir baltažiedžio vairenio rboh baltymai buvo sugrupuoti į keturias grupes atitinkančias AtrbohD, E, F ir H. Vienas identifikuotas homologas su unikalia, tik erškėtiniuose augaluose rasta seka buvo pavadintas rbohK. MdrbohD1-D3 ir MdrbohF genų raiška aptikta obels lapuose, ląstelių kultūroje ir ūgliuose augintuose in vitro. Šiuose audiniuose MdrbohE2 ir MdrbohH1-H2 genų raiška buvo skirtinga. Skirtingoms obels ūglių augimo stadijoms buvo būdingi MdrbohD1-D2 ir MdrbohF genų raiškos skirtumai, kas leidžia daryti prielaidą apie transkripcinę Mdrboh genų reguliaciją obels ūgliuose.Genes encoding rboh (respiratory burst oxidase homolog) have been described in a variety of plant species. Transcriptional regulation of rboh genes has been shown during plant response to stress or phytohormone treatment. In vitro conditions often induce plant stress that leads to slow plant growth and senescence. Production of reactive oxygen species is an important manifestation of plant environmental stress. Expression of rboh homologs in plant tissues grown under in vitro conditions and their role in response to abiotic stress is not fully understood. Therefore the aim of this study was to identify rboh homologues in domestic apple (Mdrboh) and to characterize their expression in different tissues and during the senescence of apple shoot culture in vitro. Similarity searches using Arabidopsis rboh (Atrboh) protein sequences revealed nine homologous rboh genes in the apple genome. Phylogenetic analysis of proteins of four Rosaceae plants and Arabidopsis revealed four groups of related sequences that were linked to AtrbohD, E, F and H. A homologue with unique sequence was annotated as rbohK in plants of the Rosaceae family. Expression of MdrbohD1-D3 and F was detected in apple leaves, in vitro cultures of shoots and cell suspension, and MdrbohE2 and MdrbohH1-H2 varied among the tissues. The MdrbohD1-D2 and F genes were differentially expressed during different stages of apple in vitro shoot culture, suggesting transcriptional regulation of the Mdrboh genes in the apple shoot culture
Analysis of the effect of endophytic bacteria on the carbonylation of tobacco (nicotiana tabacum) cell proteins
No full textNors yra daug informacijos apie endofitinius mikroorganizmus, žinios apie augalų ir endofitų molekulinės sąveikos mechanizmus ir procesus vykstančius pradiniame augalo audinių kolonizacijos etape yra nepakankamai išsamios. Todėl tyrimo tikslas in vitro modelinėje sistemoje ištirti ankstyvosios kolonizacijos etape vykstančius procesus ir endofitinių bakterijų poveikį augalui.
Darbe aprašomi tyrimai buvo atliekami naudojant paprastojo tabako Nicotiana tabacum L. ląstelių kultūrą ir šešis skirtingus endofitinių bakterijų kamienus. Tyrimo metu buvo ištirtas endofitinių bakterijų poveikis paprastojo tabako Nicotiana tabacum L. ląstelių kultūros gebėjimui kaupti aktyviuosius deguonies junginius. Šis tyrimas buvo atliktas naudojant dichlorofloresceino diacetato fluorescencijos metodą, o viena . Gauti rezultatai rodo, jog viena iš tirtų bakterijų statistiškai mažino ADJ kaupimą. Dėl teigiamo poveikio aktyvių deguonies junginių kaupimui, tolimesniems tyrimams buvo pasirinkta Bacillus Simplex NT21. bakterija.
Sekančio tyrimo metu buvo atliktas skirtingų fluorescuojančių dažų stabilumo vertinimas skirtingose temperatūrose ir pasirinkti stabiliausi ir labiausiai tinkami dažai. Atlikus dažų stabilumo vertinimą, buvo pasirinkta dvikryptės elektroforezės metodui naudoti CF647DI dažus.
Pasirinkus tinkamiausią endofitinės bakterijos kamieną ir fluorescuojančių dažų tipą, buvo atliktas šių bakterijų inokuliacijos poveikio tabako ląstelių baltymų kaupimui ir oksidacinei modifikacijai tyrimas, naudojant dvikryptės elektroforezės metodą. Gauti rezultatai rodo, jog ištirtas endofitinės bakterijos kamieno aktyvinamas ADJ kaupimas tabako ląstelėse sukelia streso atsako, branduolio rūgščių sintezės ir metabolizmo procesuose dalyvaujančių baltymų karbonilinimo pokyčius.The studies described in this paper were carried out using a cell culture of Nicotiana tabacum L. and six different strains of endophytic bacteria. Knowledge of the molecular mechanisms of plant-endophyte interactions and the processes involved in the initial stages of colonisation of plant tissues is incomplete and lacking. Therefore, the aim of this study was to investigate in an in vitro model system the processes involved in the early colonisation phase and the effects of endophytic bacteria on the plant.
The study investigated the effect of endophytic bacteria on the cell culture of N. tabacum L. cells abilities to accumulate reactive oxygen species. This study was carried out using the dichloroflorescein diacetate fluorescence method. The results show that one of the six bacteria tested showed a statistically significant increase in the accumulation of ROS and one other strain showed an inhibition.
In the following experiment, the stability of different fluorescent dyes at different temperatures was evaluated and the most stable and suitable dyes were selected. After the stability assessment, the CF647DI dye was chosen for the bidirectional electrophoresis method.
After selecting the most suitable endophytic bacterial strain and type of fluorescent dye, the effect of inoculation of these bacteria on the accumulation and oxidative modification of tobacco cell proteins was investigated by means of bidirectional electrophoresis. The results show that this endophytic bacterial strain activated by the accumulation of reactive oxygen species in tobacco cells induces changes in the stress response, the synthesis of nuclear acids, and in the carbonylation of proteins involved in metabolic processes.Gamtos mokslų fakultetas / Faculty of Natural SciencesVytauto Didžiojo universitetas / Vytautas Magnus UniversityBiologijos katedra / Department of Biolog
Proteomics analysis of interaction of domestic apple shoots and endophytic bacteria in vitro.
No full textDomestic apple (Malus × domestica Borkh) is one of the model species suitable for targetted genome analysis, resistance to diseases and reproduction and transformation studies in vitro. It is know that endophytes have a positive effect on plant growth and can help protect against biotic and abiotic stresses. However, in vitro conditions to endophyte effects on plants can become pathogenic and induce plant stress and death. Causes of the adverse effects on plants in vitro are poorly understood. In order to evaluate the effect of endophytic bacteria on the growth of „Gala“ domestic apple shoot in vitro, the shoots were inoculated with two Bacillus spp. bacterial strains Oa_4 and Da_1 and grown under controlled conditions using two different lighting conditions. Analysis of shoot weight and number of auxilary shoots after 21 days of inoculation with bacterial endophytes revealed that endophytes in all experimental groups inhibited the growth of shoots in vitro. In order to evaluate the changes in the domestic apple protein expression after inoculation with endophytes, a proteomic analysis was performed. Differencial expression of 111 proteforms were established, of which 49 were identified. Incubation with endophytes upregulated expression of the proteins involved in oxidation–reduction, defence response, protein metabolism and regulation of their function, cellular organization, and lipid metabolic processes. The largest changes in protein expression were detected for PLAT domain protein 1 (PLAT1), whitch is related to cell migration and tissue reorganization processes, and low temperature activated protein (RD21A), which is involved in responce to stress and pathogens. Conclusions: 1. After evaluation of endophytic Bacillus spp. strain Oa_4 and Da_1 effects on domestic apple shoot in vitro condition, we established that both strains had similarl inhibiting effect on the growth of shoots (from 3,4 to 4,4 times) and the formation of auxilary shoots (from 2,7 to 3,6 times), irrespective of the lighting conditions. After incubation with endophytic bacteria, no morphological lesions were detectable, therefore the inhibition of shoot growth is not a consequence of pathogenesis. 2. Proteomic analysis revealed that the incubation of apple shoot with the endophytic Bacillus spp. strains Oa_4 and Da_1 had mainly protein epression upregulating effect (109 out of 111 significant differences) and a larger effect on protein expression was observed under reduced light conditions. Incubation with Bacillus sp. strain Oa_4 had a larger effect on shoots protein expression. 3. Evaluation cell biological processes and gene functions related to the effects of endophytic bacteria on domestic apple shoots in vitro showed that incubation with Bacillus spp. bacterial strain Oa_4 and Da_1 lead to changes in oxidation–reduction, defence response, protein metabolism and their function regulation, cellular organization and lipid metabolic processes of the apple shoots cells
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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