Project Number: 5325-21420-004-00
Project Type: Appropriated

Start Date: May 19, 2008
End Date: May 18, 2013

Objective:
1)Develop new promoter elements from potato that will allow refined expression profiles (tissue and/or developmental specificity) of transgenes to improve agronomic and quality properties of dicotyledonous crop species. 2)Discover and develop new molecular tools (promoters, terminators) from fruit trees. In particular, to isolate transcriptional control elements and polyadenylation signals from plum and apple. 3)Refine down-regulation technologies to improve general applications to metabolic regulation as well as improve design characteristics of pathogen-resistance transgenes.

Approach:
Available EST, microarray and genomic DNA databases will serve as bioinformatics data sources to identify potato gene families, and specific family members, with requisite expression profiles to serve as sources of valuable transcriptional control elements. Putative elements will be isolated from a BAC library, assembled into marker-gene fusions, and function characterized in transgenic potatoes. Polyubiquitin genes from apple and plum will serve as sources of transcriptional control elements for direction of commercial levels of transgene expression in these fruit trees. Appropriate polyubiquitin genes for these control sequences will be identified using EST databases to identify specific, highly constitutively transcribed family members. The molecular source of elements will include both BAC libraries and PCR-amplification. Putative promoter elements will be fused to standard marker and delivered to collaborators at the USDA/ARS Appalachian Fruit Research Station for introduction and characterization in apple. Glycoalkaloids will be reduced in potatoes by suppression of both branches of the SGA pathway using small inverted hairpin structures of double stranded RNA-generating constructs. Small interfering RNAs (siRNAs) will be produced by these constructs specific for both the Sgt1 and Sgt2 gene family members responsible for the initial steps in each of the two SGA biosynthetic branches, resulting in gene inactivation. Suppression constructs will be tested for efficacy via standard genetic transformation, but will ultimately be adapted for intragenic transformation and eventual commercial application. Transgenic potato tubers producing siRNAs will be evaluated for transgene expression and SGA accumulation. Replacing 5325-21420-003-00D (08/2008). BL-1, certification 5/17/07.