植物学技术

In the course of map-based cloning, mutant genes are identified through linkage to a sufficiently small region of the genetic map and subsequent DNA sequencing. This process has become fairly straightforward for Arabidopsis mutations, owing to the completed genome sequence and the disc ...

Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluoresce ...

This chapter discusses the specific details of how oligonucleotide arrays originally designed for expression analysis can be used for high-density genotyping to reveal tens of thousands of single-feature polymorphisms (SFPs). Cluster of SFPs reveal potential natural deletion ...

Insertional mutagenesis has contributed to the success of forward genetics in Arabidopsis thaliana. The availability of large collections of lines mutagenized by either transposon or T-DNA insertions, in combination with the systematic sequencing of insertion/genome junct ...

In Arabidopsis, where homologous recombination is highly inefficient, insertional mutagenesis has provided a great alternative means for identifying loss-of-function mutants in genes of interest. Many collections of T-DNA and transposon-tagged plants have been generated ...

For a long time, Arabidopsis thaliana was not a favorite of cytogeneticists because of its small chromosomes. Nevertheless, most cytogenetic approaches have become applicable to this model plant during the past decade. Recently, the very small genome, the low content, and strong cluster ...

The volume of Arabidopsis information has increased enormously in recent years as a result of the sequencing of the genome and other large-scale genomic projects. Much of the data are stored in public databases, where data are organized, analyzed, and made freely accessible to the research com ...

A powerful approach for determining the biological functions of genes in an organism is to produce mutants with altered phenotypes and physiological responses. Various approaches for mutagenesis involving chemical, irradiation, and insertional methods have been developed; e ...

The study of the intraspecific natural variation existing in Arabidopsis provides a useful resource for the dissection of its genome at the functional, ecological, and evolutionary levels. A major step in these studies is the identification and location of quantitative trait loci (QTLs) ...

The mapping method detailed here is based on the multiplex polymerase chain reaction (PCR) coamplification of 32 molecular markers, using fluorescently labeled oligonucleotides as primers. For the genotyping of a single plant from a mapping population, only two simultaneous ampli ...

In planta Agrobacterium-mediated transformation using vacuum infiltration results in transgenic Arabidopsis thaliana without the use of sterile conditions or plant regeneration. Plants are grown in pots, in standard potting mix. Agrobacterium tumefaciens, carrying an ap ...

An Agrobacterium-mediated transient expression assay has been described for in vivo analysis of constitutive or inducible gene expression in Arabidopsis plants. By simple infiltration of Agrobacterium cells carrying appropriate gene constructs into Arabidopsis leaves, ...

Chloroplast genetic engineering offers a number of unique advantages, including high-level transgene expression, multigene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic e ...

Transient expression assays with protoplasts that utilize stably integrated reporter genes along with transfected effector genes provide several advantages over assays in which both the reporter gene and effector gene(s) are transfected into protoplasts. A protocol for carry ...

Fluorescent proteins (FPs) have established themselves as valuable reporter proteins in plant molecular biology. Beside general background information about spectral properties, protein structure and maturation of different commonly used FPs, this chapter provides det ...

Luciferase is the ideal reporter gene to provide temporal and spatial information on promoter activity in Arabidopsis and other eukaryotes; the noninvasive detection of luminescence and short half-life of luciferase activity allow repeated measurements of individual seedli ...

The LexA-VP16-ER (XVE) system is an estrogen-receptor-based chemical-inducible system for use in plants. Protocols are outlined for the inducible overexpression of transgenes, inducible transgene excision, inducible gene silencing, regulated in vivo cDNA generation, indu ...

Reporter proteins allow one to monitor cellular parameters that are involved in signal transduction, development, metabolic processes, and transport. There are targeting strategies available to direct the indicator protein exactly to the locale inside the organism from which in ...

RNA extraction is a routine technique in a molecular biology lab. High quality of RNA extracted from plants is a prerequisite to succeeding in subsequent experiments such as reverse transcription-polymerase chain reaction, Northern hybridization, cDNA library construction, and ...

DNA microarrays are widely used to analyze genome-wide gene expression patterns and to study genotypic variations. They are miniaturized collections of thousands of DNA fragments arrayed on a surface. Based on nucleic acid complementary binding, they serve as a tool to interrogate comp ...