Nitrogen fixation and assimilation processes are vital to the functioning of any ecosystem. Nevertheless, studying these processes using 15N-based stable isotope probing was so far limited because of technical challenges related to the relative rarity of nitrogen in nucleic acids and proteins compared to carbon, and because of its absence in lipids. However, the recent adoption of high-throughput sequencing and statistical modelling methods to SIP studies increased the sensitivity of the method and enabled overcoming some of the challenges. This chapter describes in detail how to perform DNA- and RNA-SIP using 15N.
- MeSH
- bakteriální RNA chemie genetika izolace a purifikace metabolismus MeSH
- bakterie fixující dusík genetika metabolismus MeSH
- centrifugace - gradient hustoty MeSH
- DNA bakterií chemie genetika izolace a purifikace metabolismus MeSH
- fixace dusíku genetika fyziologie MeSH
- izotopové značení metody MeSH
- izotopy dusíku metabolismus MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Careful and thoughtful experimental design is crucial to the success of any SIP experiment. This chapter discusses the essential aspects of designing a SIP experiment, focusing primarily on DNA- and RNA-SIP. The design aspects discussed here begin with considerations for carrying out the incubation, such as, the effect of choosing different stable isotopes and target biomolecules, to what degree should a labeled substrate be enriched, what concentration to use, and how long should the incubation take. Then tips and pitfalls in the technical execution of SIP are listed, including how much nucleic acids should be loaded, how many fractions to collect, and what centrifuge rotor to use. Lastly, a brief overview of the current methods for analyzing SIP data is presented, focusing on high-throughput amplicon sequencing, together with a discussion on how the choice of analysis method might affect the experimental design.
- MeSH
- analýza dat MeSH
- bakteriální RNA chemie MeSH
- deuterium analýza MeSH
- DNA bakterií chemie MeSH
- DNA metabolismus MeSH
- izotopové značení metody MeSH
- izotopy dusíku analýza MeSH
- izotopy kyslíku analýza MeSH
- izotopy uhlíku analýza MeSH
- RNA metabolismus MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- výzkumný projekt MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Upward migration of plants to barren subnival areas is occurring worldwide due to raising ambient temperatures and glacial recession. In summer 2012, the presence of six vascular plants, growing in a single patch, was recorded at an unprecedented elevation of 6150 m.a.s.l. close to the summit of Mount Shukule II in the Western Himalayas (Ladakh, India). Whilst showing multiple signs of stress, all plants have managed to establish stable growth and persist for several years. To learn about the role of microbes in the process of plant upward migration, we analysed the root-associated microbial community of the plants (three individuals from each) using microscopy and tagged amplicon sequencing. No mycorrhizae were found on the roots, implying they are of little importance to the establishment and early growth of the plants. However, all roots were associated with a complex bacterial community, with richness and diversity estimates similar or even higher than the surrounding bare soil. Both soil and root-associated communities were dominated by members of the orders Sphingomonadales and Sphingobacteriales, which are typical for hot desert soils, but were different from communities of temperate subnival soils and typical rhizosphere communities. Despite taxonomic similarity on the order level, the plants harboured a unique set of highly dominant operational taxonomic units which were not found in the bare soil. These bacteria have been likely transported with the dispersing seeds and became part of the root-associated community following germination. The results indicate that developing soils act not only as a source of inoculation to plant roots but also possibly as a sink for plant-associated bacteria.
- MeSH
- Bacteria klasifikace izolace a purifikace MeSH
- biomasa MeSH
- Brassicaceae klasifikace mikrobiologie MeSH
- DNA bakterií genetika MeSH
- DNA fungální genetika MeSH
- kořeny rostlin mikrobiologie MeSH
- lipnicovité klasifikace mikrobiologie MeSH
- mykorhiza klasifikace izolace a purifikace MeSH
- půdní mikrobiologie MeSH
- rhizosféra MeSH
- RNA ribozomální 16S genetika MeSH
- Saussurea klasifikace mikrobiologie MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Indie MeSH
