Location: Crop Improvement and Genetics Research2011 Annual Report
1a. Objectives (from AD-416)
Design and test molecular tools to better control transgene expression and integration. Identify, characterize, and demonstrate the utility of novel gene promoter elements for control of transgene expression in cereal crops. Emphasis is to be placed on promoters that provide developmental or environmental specificity to transgene expression, but are not active in the grains harvested for food or feed. Develop new recombination systems for plants that allow precise integration of DNA into targeted locations and selective removal of unwanted transgenic DNA from the genome. Make promoters and site-specific recombination systems with proven utility available to researchers in the public and private sectors.
1b. Approach (from AD-416)
Use microarray and computer analyses from in-house and collaborative studies to identify rice, barley and wheat genes that exhibit organ-specific-, pathogen- or abiotic stress-responsive expression patterns. Isolate the corresponding promoters and examine and document their ability to control expression in transgenic cereal plants. Design and build transformation vectors incorporating site-specific recombination systems designed to target predetermined integration sites in cereal genomes and to allow excision of plasmid backbone and marker genes no longer needed after transformants are identified. Optimize codons and protein targeting signals as needed for better functionality in plant cells. Transform plants with recombinase-encoding constructs and target constructs. Demonstrate site-specific excision and/or integration reactions in plant cells.
3. Progress Report
This is the first year of a 3-year bridging project from NP302 to NP301. Research characterizing several novel gene promoters active only in specific plant organs continued. Three rice promoters that confer pollen-specific expression in rice and Arabidopsis plants have been thoroughly characterized. In addition, a rice light-responsive leaf promoter and a rice root-specific promoter were tested in transgenic plants of Brachypodium distachyon, a useful grass species that serves as a model for cereal crops. Also, three pathogen and/or stress responsive candidate genes from wheat were identified and the corresponding upstream promoter sequences were isolated. These candidate promoters are currently being used to create constructs that will be used for wheat and Brachypodium transformation in the future. Research has also continued to develop site-specific recombination systems useful in plants. The functionality of three recombinases (BxbI, ParA, PhiC31) in the reproductive cells of transgenic Arabidopsis plants was previously demonstrated, showing that the enzymes can precisely excise DNA between their target sequences from plant chromosomes, thus preventing transmission of unwanted DNA to the progeny. More recently CinH has been added to the list of plant-functional recombinase enzymes by proving its ability to function in tobacco. To investigate recombinase utility in monocot plants, constructs encoding the Bxb1 recombinase were transformed into wheat and Brachypodium. Site-specific excision mediated by Bxb1 was demonstrated in the leaves of transgenic wheat plants. While the original strategy for detection of excision events in transient assays did not prove to be dependable, several modifications were applied until a reliable 2-day assay for quantitative estimates of recombinase enzyme activity in plants was achieved. This year, a series of assays was conducted to directly compare various recombinases for excision efficiencies in onion cells. The comparisons included the well-characterized Cre recombinase and determined the efficiencies of recombinases Flp and R as well as of the wild type and enhanced (for plant expression) versions of our newly characterized recombinases - U153, ParA, Bxb1 and phiC31. Also a transient integration activity assay has been developed that will allow testing of improvements to chromosome targeting in crop genomes. This system is currently being optimized.
Thomson, J.G., Cook, M.A., Guttman, M.E., Smith, J.D., Thilmony, R.L. 2011. Novel sull binary vectors enable an inexpensive foliar selection method in Arabidopsis. BMC Research Notes. 4(44) Available: http://www.biomedcentral.com/1756-0500/4/44.