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Protein Targeting Across and into Chloroplast Membranes
Time of Update: 2020-11-19
We have described in this chapter the methods used in our laboratory for investigations of the import of nuclear-encoded proteins across the chloroplast envelope membranes, and for their further delivery into or across the thylakoid membrane by one of the four distinct pathways.
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Identification of DNA-Binding Proteins and Protein-Protein Interactions by Yeast One-Hybrid and Yeast Two-Hybrid Screen
Time of Update: 2020-11-19
The activation of genes is mediated by well-coordinated protein–protein interactions between transcription factors and a various number of cofactors.
In order to address the involved protein–DNA and protein–protein interactions, a number of methods have been developed that efficiently addresscis -element interacting partners.
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Quantification of Stable Isotope Label in Metabolites via Mass Spectrometry
Time of Update: 2020-11-19
CORRECTOR facilitates and speeds up the routine quantification of experimentally introduced isotope label from multiple mass spectral readouts, which are generated by routine metabolite profiling when combined with stable isotope labelling experiments.
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Functional Characterization of Water-Deficit Stress Responsive Genes Using RNAi
Time of Update: 2020-11-19
Understanding the functional role of water-deficit stress-responsive genes is important in order to develop stress-tolerant plants.
Further, precautions that should be taken while developing RNAi lines and during stress imposition are discussed.
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Analysis of Rice Proteins Using SDS-PAGE Shotgun Proteomics
Time of Update: 2020-11-19
In this chapter we describe the workflow used in our laboratory to analyze rice leaf samples using label-free shotgun proteomics based onSDS -PAGE fractionation of proteins.
Rice proteomics has benefitted substantially from successful execution of shotgun proteomics techniques.
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Alkaloid Accumulation in Catharanthus roseus Suspension Cultures
Time of Update: 2020-11-19
A number of these secondary products are very valuable, such as taxol (4 ), and plant cell culture may provide an additional source of supply if: •There is a high demand for the product.
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Mushroom (Agaricus bisporus)
Time of Update: 2020-11-19
tumefaciens for 3 h with acetosyringone, a signaling molecule that launches the gene transfer mechanism, co-cultivation of the induced bacterium and gill explants for 3 d, and selection for transformants based on an inherited resistance to the antibiotic hygromycin.
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Identification of Stress-Responsive Genes in Plants Using Suppression Subtraction Hybridization: Ozone Stress as an Example
Time of Update: 2020-11-19
The popularity of this technique stems from the ease of conducting this procedure in any laboratory set up for basic molecular biology research.
Further, the availability of a comprehensive kit for conducting suppression subtractions from BD Biosciences has made this technique easy to adapt and adopt to any biological system.
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ThiolDisulfide Redox Proteomics in Plant Research
Time of Update: 2020-11-19
Abiotic stresses often cause metabolic imbalances which affect cellular redox homeostasis and alter the rate of reduction state of functional and regulatory protein thiols and the rate of reactive oxygen species release.
The understanding of the cell response to progressive stress must include knowledge on the thiol redox state of specific proteins.
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Carlavirus Isolation and RNA Extraction
Time of Update: 2020-11-19
Carlaviruses are noted for their narrow host range and tendency to induce little or no symptoms.
This has led to many of the common names of carlaviruses, including carnation latent (CLV), American hop latent (AHLV), and lily symptomless virus (LSV).
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Medicago truncatula Proteomics for Systems Biology: Novel Rapid Shotgun LC-MS Approach for Relative Quantification Based on Full-Scan Selective Peptide Extraction (Selpex)
Time of Update: 2020-11-19
In plant systems biology, several comparative studies have been carried out using liquid chromatography shotgun mass spectrometry (LC-MS/MS) and database-dependent protein identification analyses in combination with the spectral count for relative quantification.
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Microspore Culture in Brassica
Time of Update: 2020-11-19
Pioneering research in Brassica microspore culture ( 1 , 2 , 3 )rapidly led to the realization that microspores provide a powerful alternative to protoplast culture as a single-celled culture method in plants.
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Apple (Malus domestica)
Time of Update: 2020-11-19
Transformation is a key to sustaining this demand—permitting the potential enhancement of existing cultivars as well as to investigate the development of new cultivars resistant to pest, disease, and storage problems that occur in the major production areas.
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Xylem Sap Proteomics
Time of Update: 2020-11-19
Unlike cell compartments which are amenable to purification in sufficient amount prior to proteomic analysis, the xylem sap has to be collected in particular conditions to avoid contamination by intracellular proteins and to obtain enough material.
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Isolation of Genetic Material from Arabidopsis Seeds
Time of Update: 2020-11-19
Here, we describe a series of methods suitable for the reproducible and abundant isolation of total RNA, genomicDNA , and total protein from dry or imbibedArabidopsis seeds. The resulting material i
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Virus-Induced Gene Silencing as a Reverse Genetics Tool to Study Gene Function
Time of Update: 2020-11-19
VIGS is especially useful for plants that are recalcitrant for transformation and for genes that cause embryo lethality.
VIGS facilitates a rapid comparison of knockdown phenotypes of the same gene in different breeding lines or mutant backgrounds, as the same vector is easily inoculated into different plants.
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Organophosphorus Hydrolase: A Multifaceted Plant Genetic Marker Which Is Selectable, Scorable, and Quantifiable in Whole Seed
Time of Update: 2020-11-19
Organophosphorus hydrolase (OPH, EC 3.1.8.1) provides a novel function as an alternative genetic marker system for use in many types of plant transformations.
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Reverse Genetics in Medicago truncatula Using Tnt1 Insertion Mutants
Time of Update: 2020-11-19
truncatula genome sequencing, availability of large-scale mutant populations becomes a priority.
Therefore, this PCR-based reverse screening is a rapid way of identifying knock-out mutants for specific genes inTnt1 -tagged population ofM.
One can search the database to find an insertion line for the gene of interest.
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RNA Fractionation by Density Gradient Centrifugation
Time of Update: 2020-11-19
Isopycnic ultracentrifugation of virus preparations in caesium chloride (or similar density medium) gradients separates the viral components according to their buoyant density.
Once the density gradient medium is removed, the RNA is extracted by protocols applicable to each particular virus.
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Micropropagation Systems of Feijoa (Acca sellowiana (O. Berg) Burret)
Time of Update: 2020-11-19
The conventional clonal propagation methods of this species, such as cutting and grafting, have shown low efficiency.
Therefore, tissue culture techniques were developed for mass propagation.
Additional techniques including synthetic seed technology and temporary immersion system are also described.
