|Frederico, Maria - UNIV OF WISCONSIN|
|Kaeppler, Heidi - UNIV OF WISCONSIN|
Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 25, 2004
Publication Date: January 15, 2005
Citation: Frederico, M., Kaeppler, H., Skadsen, R.W. 2005. A minimal promoter of a novel stress-responsive ltp drives progressive gfp expression during barley spike development, embryogeneis and germination. Plant Molecular Biology. 57:35-51. Interpretive Summary: The economy of the upper Midwest region continues to be harmed by Fusarium graminearum, a fungal pathogen that causes scab disease of barley and wheat. Adequate genetically resistant sources of barley and wheat do not exist, so it may be necessary to create resistance by putting antifungal proteins into the fungus’ preferred route of infection, the relatively soft tissue surrounding the seed, known as the pericarp epithelium. To obtain such a level of specific targeting of antifungal protein synthesis, it is first necessary to develop a gene promoter to direct this synthesis. We have discovered a gene that is turned on mainly in the pericarp epithelium and is totally turned off in leaves. We obtained the promoter for this gene (Ltp6) and showed that it turned on the production of a reporter gene protein that we attached to it. With this result, we can now attach a gene for an antifungal protein that is found in barley seeds and go on to produce barley that should have resistance to this pathogen. This gene promoter, and others that we are developing, are the only tissue-specific ones available for barley. These will also be used by many other barley and wheat labs attempting to solve the Fusarium problem. This research will be of benefit to barley and wheat producers, maltsters and brewing companies.
Technical Abstract: The search for a gene promoter to drive preferential transgene expression in the pericarp epidermis of developing barley (Hordeum vulgare L.) seeds resulted in the cloning and analysis of a novel barley gene. Identification of a transcript highly expressed in the pericarp epidermis but not in leaves was accomplished by differential display. The full sequence was cloned, and inverse PCR was used to derive 2345 bp of upstream sequence. The gene encoded a polypeptide of 124 amino acids showing 87% identity with WBP1A, a wheat lipid transfer protein (LTP), but much lower homology to other barley LTPs. In addition to the pericarp epidermis, this Ltp-like gene, designated Ltp6, is highly expressed in coleoptiles and embryos under normal growth conditions. Messenger RNA levels increase in seedling tissues during salt and cold treatments and under applied abscisic acid and salicylic acid. The tissue-specific and response patterns of Ltp6 are distinct from other known barley Ltp genes. Quantitative real time PCR was used to assess the level of transcription conferred by different promoter deletion constructs using sgfp as a reporter in transient expression assays. All constructs containing at least 192 bp of upstream sequence, and the 5’UTR, conferred tissue-specific expression and retained most of the promoter strength. Deletion of 64 bp (-192/-128) from this upstream sequence resulted in an 80% drop in expression. A minimal 247 bp Ltp6 promoter was capable of reproducing the tissue-specificity and developmental expression of this gene in transgenic barley.