Modified small-scale batch procedure for isolation of dsRNA from Cryphonectria parasitica

Cytoplasmically-transmissible viral double-stranded RNAs of the genus Hypovirus cause reduced virulence (hypovirulence) in the chestnut blight fungus Cryphonectria parasitica. Biological control of this fungal disease is done by inoculating selected artificial hypovirulent strains of C. parasitica in the wounds of attacked chestnut trees and is followed by testing the transmissibility of dsRNA to C. parasitica isolates affecting these trees. Here we present a modified protocol of isolation and detection of dsRNA. The proposed procedure requires smaller amounts of fungal material for dsRNA detection and uses less reagents, thus resulting in appreciable cost savings.Les ARN double brin (ARNdb) viraux du genre Hypovirus à transmission cytoplasmique réduisent la virulence (hypovirulence) du Cryphonectria parasitica, le champignon responsable de la brûlure du châtaigner. La lutte biologique contre cette maladie fongique se fait par l'inoculation, dans des blessures de châtaigniers atteints, de souches choisies de C. parasitica artificiellement hypovirulentes, suivie de l'analyse de la transmissibilité des ARNdb aux isolats de C. parasitica qui affectent ces arbres. Nous présentons ici un protocole modifié d'isolement et de détection d'ARNdb. La procédure proposée requiert une plus petite quantité de matériel fongique pour la détection d'ARNdb et demande moins de réactifs; il en découle donc des économies appréciables

Optimalizácia podmienok expresie droserovej chitinázy (Drosera rotundifolia L.) v troch expresných kmeňoch E. coli

Round-leaf sundew (Drosera rotundifolia L.), family Droseraceae, genus Drosera, is one of a few plant species with a strong antifungal potential. Chitinases of carnivorous plants play an important role in decomposition of chitin-containing cell structures of insect prey. The cell wall of many phytopathogenic fungi also contains chitin, which can be utilized by chitinases, thus round-leaf sundew represents an interesting gene source for plant biotechnology. The purpose of this study was to compare the suitability of 3 different E. coli expression strains (E. coli BL21- CodonPlus® (DE3)-RIPL, E. coli ArcticExpress (DE3)RIL and E. coli SHuffle® T7) for production and isolation of heterologous round-leaf sundew chitinase (DrChit). Results showed that the recombinant protein was successfully expressed in all three strains, but occurred in insoluble protein fraction. To get the DrChit protein into soluble protein fraction some modifications concerning to induction temperatures and concentration of the IPTG inductor were tested. In addition, composition of lysis buffer has been modified with supplementation of strong non-ionic detergents, Triton® X100 and Tween® 20, respectively. As these modifications didn’t increase the amount of the DrChit protein in soluble fraction, therefore, its isolation under denaturing conditions and subsequent refolding for activity assays is recommended.Rosička okrúhlolistá (Drosera rotundifolia L.), rodina Droseraceae, rod Drosera, je jednou z mála rastlinných druhov so silným antifungálnym potenciálom. Chitinázy mäsožravých rastlín zohrávajú dôležitú úlohu pri dekompozícií bunkových štruktúr obsahujúcih chitín z tela chyteného hmyzu. Bunkové steny viacerých fytopatogénnych húb obsahujú chitín, ktorý môže byť využívaný chitinázami, čo robí z rosičky okrúhlolistej zaujímavý genetický zdroj pre rastlinné biotechnológie. Cieľom práce bolo porovnanie vhodnosti použitia 3 rozdielnych expresných kmeňov E. coli (E. coli BL21-CodonPlus® (DE3)-RIPL, E. coli ArcticExpress (DE3)RIL a E. coli SHuffle® T7) pre produkciu a izoláciu heterológnej chitinázy rosičky okrúhlolistej (DrChit). Výsledky ukázali, že rekombinantný proteín bol úspešne exprimovaný vo všetkých 3 kmeňoch, ale vyskytoval sa v nerozpustnej proteínovej frakcii. Aby sa DrChit proteín dostal rozpustnej proteínovej frakcie, boli testované modifikácie teploty indukcie a koncentrácie induktora IPTG. Ďalej bolo modifikované zloženie lyzačného pufru pridaním silných neiónových detergentov, Triton® X100 a Tween® 20. Pretože tieto modifikácie nezvýšili množstvo získaného DrChit proteínu v rozpustnej frakcii, je odporúčaná jeho izolácia za denaturačných podmienok a následná obnova štruktúry proteínu

Study on metal-triggered callose deposition in roots of maize and soybean

Abstract: Callose plays important roles in a variety of processes of plant development, and/or in a response to a range of biotic and abiotic stresses. In the current work we have studied and compared the effect of lead, cadmium and arsenic on accumulation of newly formed callose deposits in the roots of maize and soybean. We observed formation of characteristic callose deposits in the root cell walls, probably associated with plasmodesmata, depending on the type of metal and the plant species investigated. Further, the callose turnover was analysed by measuring of total callose content as well as activities of total β-(1,3)-glucanases in roots. The latter enzymes are responsible for callose depletion, and their possible role during metal stress has previously been proposed. However, neither of these biochemical values appeared to be sufficiently reliable for scoring the altered callose turnover (including local deposits) in plant tissue. The microscopical observations are discussed in light of the biochemical data obtained

The current state and prospects for sustainable development of tourism in Slovakia.

Cestovní ruch přispívá k rozmachu destinací, kam přináší finanční prostředky, nová pracovní místa, vyšší úroveň služeb, či rozvoj infrastruktury. Ovšem pokud se cestovní ruch nerozvíjí udržitelně, doprovázejí ho i negativní faktory. Jde hlavně o negativní dopady na životní prostředí, jako například: znečištění vody, půdy, zvýšená hlučnost a přelidnění destinace během sezóny. Jako reakce na zhoršující se stav životního prostředí a možné neodvratitelné změny způsobené nezodpovědným lidským jednáním, nabývá na významu myšlenka udržitelného rozvoje. Teoretická část popisuje cestovní ruch na Slovensku a poukazuje na plány udržitelného rozvoje cestovního ruchu. V praktické části jsou zhodnoceny výsledky dotazníku, který se týká povědomí o udržitelném cestovním ruchu na Slovensku. Hlavním cílem diplomové práce je zhodnocení současného stavu v oblasti udržitelného cestovního ruchu a návrh možností jeho rozvoje do budoucna.Tourism contributes to the prosperity of destinations by bringing financial resources, new jobs, a higher level of services and infrastructure development. However, if tourism does not develop sustainably, it is accompanied by negative factors, as well. Especially, negative impacts on the environment, such as: water and soil contamination, increased noise and overcrowded destination during the high season. As a response to the worsening state of the environment and possible irreversible changes caused by irresponsible human behavior, the idea of sustainable development is becoming more important. The theoretical part describes tourism in the Slovak Republic and highlights plans for sustainable tourism development. In the practical part, the results of the questionnaire, related to the awareness of sustainable tourism in the Slovak Republic, are evaluated. The main objective of this thesis is to evaluate the current situation in the field of sustainable tourism and to make suggestions for its future development.Cestovný ruch prispieva k rozmachu destinácií, kam prináša finančné prostriedky, nové pracovné miesta, vyššiu úroveň služieb, či rozvoj infraštruktúry. Avšak pokiaľ sa cestovný ruch nerozvíja udržateľne, sprevádzajú ho aj negatívne faktory. Ide hlavne o negatívne dopady na životné prostredie, akými sú napríklad znečistenie vody, pôdy, zvýšená hlučnosť, či preľudnenie destinácie počas sezóny. Ako reakcia na zhoršujúci sa stav životného prostredia a možné neodvrátiteľné zmeny spôsobené nezodpovedným ľudským konaním, nadobúda na význame myšlienka udržateľného rozvoja. Teoretická časť popisuje cestovný ruch na Slovensku a poukazuje na plány udržateľného rozvoja cestovného ruchu. V praktickej časti sú zhodnotené výsledky dotazníku, ktorý sa týka povedomia o udržateľnom cestovnom ruchu na Slovensku. Hlavným cieľom diplomovej práce je zhodnotenie súčasného stavu v oblasti udržateľného cestovného ruchu a návrh možností jeho rozvoja do budúcna

Creating a Tour to a Chosen Destination (Guatemala)

Nowadays tourists have the opportunity to travel beyond the traditional countries to some exotic destinations which were difficult to access in the past. Guatemala is a country in Central America that offers many tourist attractions. The aim of this thesis is to create a package tour to Guatemala which will be unique in the market of travel agencies. The tour will be scheduled in such way that the participants will visit the most beautiful places of the country such as natural beauties and historical monuments. The tour participants will visit the popular attractions of Guatemala and also those less known. In all the destinations the participants will have enough time for exploring and relaxing

The current state and prospects for sustainable development of tourism in Slovakia.

Tourism contributes to the prosperity of destinations by bringing financial resources, new jobs, a higher level of services and infrastructure development. However, if tourism does not develop sustainably, it is accompanied by negative factors, as well. Especially, negative impacts on the environment, such as: water and soil contamination, increased noise and overcrowded destination during the high season. As a response to the worsening state of the environment and possible irreversible changes caused by irresponsible human behavior, the idea of sustainable development is becoming more important. The theoretical part describes tourism in the Slovak Republic and highlights plans for sustainable tourism development. In the practical part, the results of the questionnaire, related to the awareness of sustainable tourism in the Slovak Republic, are evaluated. The main objective of this thesis is to evaluate the current situation in the field of sustainable tourism and to make suggestions for its future development

The promiscuity of heterospecific lox sites increases dramatically in the presence of palindromic DNA

Heterospecific lox sites are mutated lox sites that in the presence of Cre recombinase recombine with themselves but not or much less with wildtype loxP. We here show that in Escherichia coli both lox511 and lox2272 sites become highly promiscuous with respect to loxP when in the presence of Cre one of the recombination partners is present in a larger stretch of an inverted repeat of non-lox DNA. In such a palindromic DNA configuration, also the occurrence of other DNA repeat-mediated recombination events is somewhat increased in the presence of Cre. The results indicate that in recombinase mediated cassette exchange or other double lox applications based on the exclusivity of heterospecific lox sites, or in research combining Cre-lox approaches with hairpin RNA for gene silencing, the presence of duplicated DNA around lox sites has to be taken into account. It is proposed that the presence of palindromic non-lox DNA interferes with the homology search of the Cre enzyme prior to the actual recombination event