1a. Objectives (from AD-416)
The objectives of this project are: 1) establish a functional and operable Tissue-Specific Transgene Removal and Containment System (TRECS) in plants through the use of enhancer-blocking insulators and PtAGIP promoter; 2) improve the established TRECS efficiency through the adoption and analysis of the identified genetic and genomic factors that influences the gene excision process in plants; and 3) evaluate TRECS' stability and efficiency under various conditions.
1b. Approach (from AD-416)
New TRECS vectors with isolated enhancer-blocking insulators and promoters will be made and introduced into tobacco plants. Gene excision efficiency will be analyzed, compared, and evaluated in transgenic lines. The genetic and genomic factors that influence gene excision efficiency will be analyzed in detail. The gene excision efficiency will be further improved by testing different nuclear-localization signals and the identified genomic (e.g. location, chromain structure and change) factors. The gene excision events will be monitored and detected by PCR amplification and Southern blot in T0 and T1 generations. The chosen plants with the desired excision efficiency will be grown under different temperature regions in controlled growth chambers for evaluation of TRECS' stability and efficiency.
3. Progress Report
The goal of this project is to further improve the Tissue-Specific Transgene Removal and Containment System (TRECS) excision efficiency in plants. To obtain a better female tissue promoter, three floral meristem-specific promoters were isolated through genome chromosome walking and verified by DNA sequencing. The isolated promoters were incorporated into TRECS to develop several new TRECS vectors, which are expected to give better excision efficiency. Plants were transformed with these new TRECS constructs. The ADODR has monitored activities through emails, meetings, and calls.