Isolation of Plant Genes by T-DNA and Transposon Mutagenesis — Gene Tagging

Abstract

Modern biology depends to a large extent on the identification, isolation and characterization of genes. Describing the expression, function and regulation of these genes is crucial in understanding many of the fundamental questions in development, morphology, pathogen resistance, stress responses, and other phenomena. The classical approaches to cloning require information about the gene product, i.e., the transcript or polypeptide. This information can be used to identify cDNA or genomic clones from a DNA library employing a variety of strategies including different hybridization procedures, synthetic oligonucleotides, expression vectors, specific antibodies or heterologous DNA probes, etc. However, the majority of genesis not easily cloned with these techniques. For most of the agronomically important plant genes very limited information is available about their polypeptide products. Often the only character at hand is a phenotype, a feature difficult to utilize in most of the cloning techniques listed above. Here, gene tagging offers an attractive alternative. The rationale for gene tagging as a technique to identify and isolate genes is that random or directed insertion of DNA sequences into a gene both mutates and tags the gene. The insertional element in plant systems is either T-DNA or transposons (or a combination thereoff). T-DNA and transposon-mediated gene tagging have been used successfully in the isolation of several plant genes in Arabidopsis, snapdragon (Antirrhinum majus) and maize (Zea mays) genes, respectively. In this review we present the basic techniques and results using the gene tagging approach for gene isolation.