Poster: Transcription Regulation

Abs # 1062: Identification and characterization of promoter elements controlling the expression of vegetative storage proteins from alfalfa (Medicago sativa L.)

Presenter:	Sors, Thomas G, tsors@purdue.edu
Authors	Sors, Thomas G (A)   Cunningham, Suzanne M (A)   Volenec, Jeffrey J (A)
Affiliations:	(A): Department of Agronomy / Purdue University

In alfalfa (Medicago sativa L.), vegetative storage proteins (VSPs) in taproots function as an N source used for initial shoot growth during spring and shoot regrowth after defoliation. Our objective was to isolate and characterize VSP promoters from alfalfa. VSPs accumulate only in taproot tissues of perennial Medicago species, with high VSP deposition rates occurring in autumn as plants cold acclimate. The high molecular weight (hmw; 32 kDa) and medium molecular weight (mmw; 19 kDa) VSPs share high protein sequence homology to Type III acidic endochitinases. A 30 amino acid signal peptide encoded at the 5' end of the hmw-vsp is predicted to direct protein accumulation into the vacuole of taproot parenchyma cells. To verify vacuolar-targeted protein accumulation, signal peptide/GFP protein fusion constructs will be used. An adaptor PCR-based technique was used to "walk" upstream from the coding region and isolate the VSP promoters. Gel-mobility shift assays and DNase I footprinting analysis will be used to identify potential cis-acting elements. Upon identification of the putative regulatory sequences, promoter deletion analyses will be performed using the green fluorescent protein (GFP) as a reporter of gene expression. The project is designed to identify cis-regulatory elements required for developmental and tissue-specific gene expression in alfalfa taproots. Promoter sequences that are responsive to defoliation, cold acclimation, N nutrition including symbiosis, pathogen attack, and methyl jasmonate application will be studied. By using transgenic alfalfa reporting VSP expression in field and growth-chamber experiments, we hope to gain a better understanding of VSP gene expression and their involvement in alfalfa persistence.