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JXB Advance Access published online on July 12, 2005
Journal of Experimental Botany, doi:10.1093/jxb/eri242
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
Received January 12, 2005
Accepted June 4, 2005

RESEARCH PAPER

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

Biljana Stangeland 1, Ragnhild Nestestog 2, Paul E. Grini 3, Nirma Skrbo 3, Anita Berg 3, Zhian Salehian 2, Abul Mandal 4, and Reidunn B. Aalen 3*

1 Plant Molecular Biology Laboratory, Department of Plant and Environmental Sciences, University of Life Sciences, PO Box 5003, N-1432 Ås, Norway; Present address: The National Hospital, Section for Gene Therapy, N-0027 Oslo, Norway
2 Plant Molecular Biology Laboratory, Department of Plant and Environmental Sciences, University of Life Sciences, PO Box 5003, N-1432 Ås, Norway
3 Department of Molecular Biosciences, University of Oslo, Oslo, PO Box 1041 Blindern, N-0316 Oslo, Norway
4 School of Life Sciences, University of Skövde, SE-54128 Skövde, Sweden

* To whom correspondence should be addressed.
Reidunn B. Aalen, E-mail: reidunn.aalen{at}imbv.uio.no


  Abstract

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.

Keywords: Embryo; endosperm; GUS; promoter trapping.
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