Identification and Characterization of a T-DNA Promoter Trap Line of Arabidopsis thaliana Uncovers an Embryo Sac-Specific Bi-directional Promoter

Abstract

The Arabidopsis thaliana promoter trap line GUS-650 showing uidA reporter gene expression specifically in mature embryo sacs was found to carry transfer DNA (T-DNA) insertion in the 5′ untranslated region (UTR) of At3g17150. At3g17140 and At3g17150 are a pair of head-to-head-oriented genes separated by a 668-bp intergenic region encoding for a putative pectin methyl esterase inhibitor protein. In the GUS-650 line, uidA reporter gene was inserted in head-to-head orientation to the At3g17140 gene. Presence of At3g17140 transcripts in the GUS-650 line indicated that T-DNA insertion did not affect this gene and the 668-bp intergenic region functions as a bi-directional promoter. The 672-bp fragment separating the uidA and At3g17140 genes was cloned and placed in forward or reverse orientation next to the uidA gene. Transgenic Arabidopsis plants carrying either of these constructs showed β-glucuronidase (GUS) activity in the ovules confirming that the 672-bp sequence is a bi-directional promoter specific to ovules. These results were also supported by the presence of cis-acting motifs such as TATA box, CAAT box, seed storage protein element (CAACAC), endosperm-specific elements (AACA), Skn-1 motif (GTCAT) and bZIP transcription factor binding element (ACGT) within the 672-bp sequence in both orientations. The significance of a promoter trap approach and the utility of such a promoter are discussed.