T-DNA Mediated Gene Tagging in Arabidopsis

Abstract

One of the major questions in biology is concerned with how individual genes within an organism are co-ordinately expressed to elicit the complex morphological and developmental processes which occur during the life cycle. In order to answer this central question, the genes which play determinative roles in the regulation of these processes must be isolated and characterized. The transcripts and encoded by these genes are present at relatively low abundances to specific cell at precise stages of development. makes the cloning of these genes conventional methods (e .g . differential cDNA library screening or screening) problematic . At there are two strategies which are to be extremely useful this area of developmental biology. The first is mutant analysis and the second is gene C"lSiSH!iS'In this chapter we will describe in detail the T-DNA tagging approach that we have adopted in our laboratory as a means to identify and isolate genes which are expressed differentially during plant development. We will describe the T-DNA­ based vector which we have constructed and discuss its merits as both an insertional mutagen and a promoter trap. Several genes have been isolated in other laboratories using T-DNA insertional mutagenesis and we will review the progress in this area to date. Over recent years Arabidopsis thaliana has been adopted as one of the major dicot species to be used as a model for studies in plant gene expression and dcvel­ opment, and we will also discuss its relative merits . The success of the T-DNA tagging IS on an efficient means of transformants and we therefore describe in detail a rapid and efficient method of Agrobaclcriul1Hllcdiated transformation of Arabidopsis which we have developed in our Procedures for the screening of transgenic for putative mutants and gene fusion expression will then be described . The promoter vector we employ contains a promotcrless gene, the activity of which can be assayed both fluorimetrically and histochemically, and detailed protocols will be presented for both procedures . Methods for determining the num­ ber of loci and precise copy number of the T-DNA vector within an individual linewill be described. Finally, we will describe a method for the cloning of tagged genes using inverse PCR (IPCR).