植物生理

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of ...

Restriction fragment length polymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows ...

Epigenetic changes in the plant genome are associated with differential genome methylation, histone modifications, and the binding of various chromatin-binding factors. Methylation of cytosine residues is one of the most versatile mechanisms of epigenetic regulation. The an ...

Methylation is a reversible covalent chemical modification of DNA intended to regulate gene expression, genome stability, and chromatin structure. Although there are various methods of methylation analysis, most of them are either laborious or expensive, or both. Here, we describe a q ...

DNA methylation is a major mechanism for the reversible control of gene expression, chromatin structure, and genome stability. Methylation analysis at a given locus allows one to evaluate levels of chromatin packaging, gene expression, and even homologous recombination. We have shown ...

Amplifying and sequencing DNA after bisulfite treatment of genomic DNA reveals the methylation state of cytosine residues at the highest resolution possible. However, a thorough analysis is required for statistical evaluation of methylation at all sites in each genomic region. Sev ...

Methylation of cytosines is a very important epigenetic modification of genomic DNA in many different eukaryotes, and it is frequently involved in transcriptional regulation of genes. In plants, DNA methylation is regulated by a complex interplay between several methylating and de ...

Epigenetic effects such as gene silencing and variable expression are unintended consequences of plant transformation, a problem that is present in the transformation of all plant species. There is not yet a reliable way to prevent epigenetic silencing; however, the probability of epig ...

Homologous recombination is a double-strand break repair mechanism operating in somatic cells and involved in meiotic crossovers in plants. It is responsible for the maintenance of genome stability and thus plays a crucial role in adaptation to stress. Recombination between homolo ...

DNA double strand breaks (DSBs) arise from spontaneous DNA damage due to metabolic activities or from direct and indirect damaging effects of stress. DSBs are also formed transiently during such processes as replication, transcription, and DNA repair. The level of DSBs positively corre ...

Virus-induced gene silencing (VIGS) exploits endogenous plant antiviral defense mechanisms to posttranscriptionally silence the expression of targeted plant genes. VIGS is quick and relatively easy to perform and therefore serves as a powerful tool for high-throughput funct ...

Epigenetic gene regulation via histone modifications controls different processes ranging from embryonic development, vegetative development, floral induction, floral organ development, to pollen tube growth. The identification of an increasing number of epigeneti ...

Genome-wide analysis of histone modifications via ChIP-chip (chromatin immunoprecipitation followed by whole genome tiling array hybridization) may generate lists of up to several thousand potential target genes. In the case of the model organism Arabidopsis thaliana, sever ...

Genome-wide targets of chromatin-associated factors can be identified by a combination of chromatin-immunoprecipitation and oligonucleotide microarray hybridization. Genome-wide mircoarray data analysis represents a major challenge for the experimental biolo ...

Chromatin immunoprecipitation in combination with DNA-microarray hybridization (ChIP-chip) allows the identification of chromatin regions that are associated with modified forms of histones on a genomic scale. The ChIP-chip workflow consists of the following steps: gener ...

In plants, miRNA bind to target RNAs with a high degree of complementarity. In this chapter, a simple method for computationally predicting plant miRNA targets, using a position-dependent scoring system, is described.

For the experimental analysis of miRNAs and other small RNAs in the 20–25 nucleotide (nt) size range, the first and most important step is the isolation of high-quality total RNA. Because RNA degradation products can mask or dilute the presence of true miRNAs, it is important when choosing a method that ...

This chapter presents procedures for the computational identification of plant miRNA genes. In the first procedure, homologs of known miRNAs are identified in a database of genomic or cDNA sequence. In the second procedure, previously unidentified miRNA families are predicted throu ...

MicroRNAs (miRNAs) are an important class of endogenous small silencing RNAs in both plants and animals. They regulate the expression of a wide range of target genes that are involved in many important biological processes. Biogenesis of plant miRNAs requires a distinct set of proteins, incl ...

Transient assays provide a convenient alternative to stable transformation. For small RNA analysis in plants, the most widely used method, commonly named agroinfiltration, makes use of Agrobacterium tumefaciens to deliver transgenes into leaf cells of Nicotiana benthamiana. C ...