植物学技术

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 ...

Argonaute (AGO) proteins recruit small RNAs to form effector complexes of RNA interference (RNAi), collectively termed RNA-induced silencing complexes (RISCs). Here, we describe detailed protocols for the purification of AGO complexes and their associated small RNAs, using Arab ...

Plant microRNA (miRNA) processing requires at least two cleavage steps of respective precursors. The first cleavage step is from pri-miRNA to pre-miRNA, and the second cleavage step is from pre-miRNA to mature miRNA. Using northern blot analysis, we previously showed that the RNase III enzyme ...

MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs asso ...

The methods described herein first highlight the strategies that were used to discover a biotic stress-associated miRNA. This involved (1) the selection of transcripts that were more abundant in transgenic plants expressing viral-derived suppressors of RNA silencing and transcr ...

Short, interfering RNAs (siRNAs) arise from the processing of long double-stranded RNA (dsRNA) by Dicer enzymes. Dicers generate siRNA duplexes by successive hydrolysis of both strands of the dsRNA phosphodiester backbone at positions determined by measuring 21–24 nucleotides fr ...

The characterization of gene function typically includes a detailed analysis of loss-of-function alleles. In model plants, such as Arabidopsis thaliana and rice, sequence-indexed insertion collections provide a large resource of potential null alleles that can often be easily ac ...

Next-generation sequencing technologies have a substantial impact on a broad range of biological applications. Like many other groups, we use these new technologies, especially SBS (Sequence-By-Synthesis), for deep profiling of small RNA molecules in plants. Small RNAs are 21–24 nuc ...

A spatial and temporal analysis of miRNA accumulation by in situ analyses is the prerequisite of understanding the precise biological functions of miRNAs. Since miRNAs are very short molecules, their in situ analysis is technically demanding. Here, we describe a protocol for miRNA in situ de ...

This chapter describes a stepwise protocol for Agrobacterium-mediated sorghum genetic transformation. Immature embryos from sorghum plants were used as the target explants. The Agrobacterium strain LBA4404, carrying a “super-binary” vector, was used in this protocol. Agroba ...

This chapter describes a method for rapid gene expression assays in Arabidopsis. Three distinct plasmids are codelivered into leaves by microparticle bombarment. The first one carries a transacting factor gene driven by the cauliflower mosaic virus (CaMV) 35S promoter. The second pla ...

Large DNA molecules (l00 kb) are extremely sensitive to mechanical shearing m aqueous solution. Therefore, classical DNA extractron procedures from living tissues generally do not allow recovery of DNA fragments larger than 50‐100 kb m size. In recent years, technical improvements have ...

Abstract The importance of determining spatial distribution and expression of biomolecules in living organisms has been increasingly recognized because of the relevance to biological function. High resolution spatial information at the level of the cell or organism can be obtai ...

The science of plant breeding has been a major force in the development of new and improved cultivars and hybrids of major agricultural crops. All people have benefited from these developments. In conventional plant breeding, one must usually find the trait of interest within the same or closely ...

Gene overexpression as a means to determine plant gene function has been used almost since the first plant transformation protocols became viable. The goal of these experiments, as in classical genetic experiments, is to observe any phenotypic change associated with changing the expre ...