植物学技术

Genome walking is a method for determining the DNA sequence of unknown genomic regions flanking a region of known DNA sequence. The Genome walking has the potential to capture 6–7 kb of sequence in a single round. Ideal for identifying gene promoter regions where only the coding region. Genome walki ...

Next-generation sequencing has resulted in a massive flow of new information predicting the existence of many new genes, their putative promoters, as well as long and small noncoding RNA. However, this is currently largely unmatched by functional studies. A cost-effective and high-thro ...

Ligation reactions to ligate a desired DNA fragment into a vector can be challenging to beginners and especially if the amount of the insert is limiting. Although additives known as crowding agents, such as PEG 8000, added to the ligation mixes can increase the success one has with ligation reactio ...

Carbon fluxes in plants have been subject to many modeling studies. The conceptual framework of models of carbon acquisition, allocation, and metabolism in plants are first introduced, together with methods to calibrate and evaluate the validity of the resulting models. The possibili ...

In order to highlight our understanding on ecosystems functioning and resource sharing/competition, either in artificial environment or agrosystems, according to changes in the climatic conditions, it is necessary to measure accurately element fluxes within plants. Stable i ...

Considerable progress has been made in plant genome-scale metabolic reconstruction and modeling in recent years. Such reconstructions made it possible to explore metabolic phenotypes through appropriate model formulation and optimization methods. As a result, plant genome ...

Flux variability analysis enables comprehensive exploration of alternate optimal routes in a metabolic network. This method is especially useful with models such as bna572 for the developing oilseed rape embryo which is highly compartmentalized. Here, we describe a protocol for ca ...

The analysis of plant metabolic networks essentially contributes to the understanding of the efficiency of plant systems in terms of their biotechnological usage. Metabolic fluxes are determined by biochemical parameters such as metabolite concentrations as well as enzyme pro ...

This chapter describes a practical procedure to dissect metabolic systems, simplify them, and use or derive enzyme rate equations in order to build a mathematical model of a metabolic system and run simulations. We first deal with a simple example, modeling a single enzyme that follows Michael ...

The evaluation of enzyme activities, especially their capacities, represents an important step towards the modelling of biochemical pathways in living organisms. The implementation of microplate technology enables the determination of up to 50 enzymes in relatively large num ...

In this chapter we illustrate the methodology for high-throughput metabolic flux analysis. Central to this is developing an end to end data pipeline, crucial for integrating the wet lab experiments and analytics, combining hardware and software automation, and standardizing data re ...

An important aspect of kinetic modeling is the ability to provide predictive information on network control and dynamic responses to genetic or environmental perturbations based on innate enzyme kinetics. In a top-down approach to model assembly, unknown kinetic parameters are cal ...

Metabolic flux analysis (MFA) is a powerful approach for quantifying plant central carbon metabolism based upon a combination of extracellular flux measurements and intracellular isotope labeling measurements. In this chapter, we present the method of isotopically nonstati ...

Metabolic flux analysis (MFA) is a powerful tool for exploring and quantifying carbon traffic in metabolic networks. Accurate flux quantification requires (1) high-quality isotopomer measurements, usually of biomass components including proteinogenic/free amino acids or ...

This chapter describes a procedure to analyze 13C-labeled phosphorylated compounds by liquid chromatography tandem mass spectrometry. Phosphorylated compounds, intermediaries of the glycolysis and pentose phosphate pathway, are separated by anion exchange chromato ...

We describe an approach to extract 13C-labeled sugars (glucose, fructose, maltose, sucrose, myo-inositol as well as glucose from starch) from plant tissues and to analyze their isotopomer distribution by gas chromatography–mass spectrometry (GC-MS). Sugars are derivatized with N, ...

Plant metabolic pathways and the molecular and atomic fluxes through them can be deduced using stable isotopically labeled substrates. To this end one prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of ...

Comprehensive analysis of isotopic labeling patterns of metabolites in proteinogenic amino acids and starch for plant systems lay in the powerful tool of 2-Dimensional Nuclear Magnetic Resonance (2D NMR) spectroscopy. From 13C-labeling experiments, 2D NMR provides informati ...

The analysis of stable isotope incorporation following feeding of 13C-labeled precursors to plant tissues provides the constraints necessary for metabolic flux analysis. This protocol describes the use of one-dimensional 1H and 13C nuclear magnetic resonance spectroscopy f ...

While steady-state 13C metabolic flux analysis is a powerful method for deducing multiple fluxes in the central metabolic network of heterotrophic and mixotrophic plant tissues, it is also time-consuming and technically challenging. Key steps in the design and interpretation of ste ...