Molecular analysis of Arabidopsis endosperm and embryo promoter trap lines: reporter-gene expression can result from T-DNA insertions in antisense orientation, in introns and in intergenic regions, in addition to sense insertion at the 5' end of genes.
Random insertions of promoterless reporter genes in genomes are a common tool for identifying marker lines with tissue-specific expression patterns. Such lines are assumed to reflect the activity of endogenous promoters and should facilitate the cloning of genes expressed in the corresponding tissues. To identify genes active in seed organs, plant DNA flanking T-DNA insertions (T-DNAs) have been cloned in 16 Arabidopsis thaliana GUS-reporter lines. T-DNAs were found in proximal promoter regions, 5' UTR or intron with GUS in the same (sense) orientation as the tagged gene, but contrary to expectations also in inverted orientation in the 5' end of genes or in intergenic regions. RT-PCR, northern analysis, and data on expression patterns of tagged genes, compared with the expression pattern of the reporter lines, suggest that the expression pattern of a reporter gene will reflect the pattern of a tagged gene when inserted in sense orientation in the 5' UTR or intron. When inserted in the promoter region, the reporter-gene expression patterns may be restricted compared with the endogenous gene. Among the trapped genes, the previously described nitrate transporter gene AtNRT1.1, the cyclophilin gene ROC3, and the histone deacetylase gene AtHD2C were found. Reporter-gene expression when positioned in antisense orientation, for example, in the SLEEPY1 gene, is indicative of antisense expression of the tagged gene. For T-DNAs found in intergenic regions, it is suggested that the reporter gene is transcribed from cryptic promoters or promoters of as yet unannotated genes.
Stangeland
Biljana
B
Plant Molecular Biology Laboratory, Department of Plant and Environmental Sciences, University of Life Sciences, PO Box 5003, N-1432 As, Norway.
Nestestog
Ragnhild
R
Grini
Paul E
PE
Skrbo
Nirma
N
Berg
Anita
A
Salehian
Zhian
Z
Mandal
Abul
A
Aalen
Reidunn B
RB
D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development; D017931:DNA Primers; D016373:DNA, Antisense / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D017930:Genes, Reporter; D007438:Introns; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014158:Transcription, Genetic
D017931:DNA Primers; D016373:DNA, Antisense; D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA