Identification of molecular markers of embryogenesis in Arabidopsis thaliana by promoter trapping

Abstract

SummaryThe technique of promoter trapping has been exploited to identify markers of embryogenesis in Arabidopsis thallana. A population of transgenic A. thallana was generated containing the promoter trap vector pAgusBin19, following Agrobacferium tumefaciens-medlated transformation. This vector contains, at the T-DNA left border, a promoterless gusA gene, which is activeted following integration downstream of a native gene promoter that directs transgene transcription. Screening of a population of 430 independent transgenic lines revealed that 74 lines (17.2%) exhibited GUS activity in siliques, as determined by fluorimetric amy. Hietochemical GUS analysis was used to identify lines that expressed GUS in embryos, and three lines that were demonstrated to contain single T-DNA Inserts were analysed in detail. Each line showed a distinct pattern of GUS fusion activity. Fusion transcripts were identified, demonstrating that transcription was initiated in the genomic DNA flanking the T-DNA left border. Inverse PCR was used to clone the T-DNA flanking sequences, and for one line a corresponding cDNA was Identified, demonetrating that the tagged sequences are transcribed. The markers represent the earliest embryonic genes known to be expressed in plants.

EXORDIUM– a gene expressed in proliferating cells and with a role in meristem function, identified by promoter trapping in Arabidopsis

“…Previous studies showed that promoter trap GUS activity in the promoter trap line AtEM201 is constitutive throughout the four cell-stage, globular and heart-stage embryo ( Figure 1a; Topping et al, 1994). GUS activity in the mature embryo is strongest in the developing radicle, with weaker staining in the cotyledons.…”

“…Previous T-DNA segregation and Southern analysis demonstrated that the promoter trap line AtEM201 contains a single copy T-DNA (Topping et al, 1994). Inverse PCR (IPCR) was used to generate 464 bp of T-DNA flanking sequence, presumed to contain part of the tagged EXO gene.…”

“…The transgenic line AtEM201 (A. thaliana ecotype C24) contains the promoter trap pDgusBin19 (Topping et al, 1991(Topping et al, , 1994. Aux1-7 and axr1-12 mutants were obtained from the Nottingham Arabidopsis Stock Centre.…”

“…Primary PCR products were cloned in the vector pGEM-T (Promega). Genomic DNA flanking the promoter trap T-DNA was isolated by inverse PCR amplification (IPCR), cloned and sequenced as described (Topping et al, 1994). A c. 1-kbp EcoRI-ClaI fragment of the IPCR product was used as a probe to screen a WT genomic library in lGEM11 (Dr Ronald Davis, Stanford; provided by Dr Jeff Dangl, formerly of the EC-…”

“…In order to identify new genes expressed in dividing plant cells, a system of promoter trap insertional mutagenesis was employed in the model experimental organism Arabidopsis thaliana (Casson et al, 2002;Lindsey et al, 1993Lindsey et al, , 1998Topping et al, 1991Topping et al, , 1994. This system allows an approach to functional genomics that relies not merely on the generation of a mutant phenotype, upon which other mutagenesis strategies rely, but also facilitates the detection of genes by the activation of a promoter-less reporter gene by the tagged gene promoter.…”