2011 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.
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.
More efficient excision tools for plant chromosome engineering. The adoption of genetically engineered crop plants has met with some consumer concerns about the presence of selectable marker genes (e.g., antibiotic resistance) in transgenic plants. To address these concerns and to provide new tools for biotechnology, ARS research scientists in the Crop Improvement/Utilization Research Unit in Albany, CA, optimized two prokaryotic recombinase enzymes for excision of marker genes from plant chromosomes. In onion cells, the improved versions of recombinases Bxb1 and ParA exhibited 10- and 12-fold increases in excision efficiencies, respectively, over those of the unmodified forms. In parallel, recombinases ParA and CinH were shown to function in the cells of tobacco and poplar trees. These novel recombination systems will help alleviate public concerns by providing the biotechnology industry convenient options for removal of unwanted DNA and precise genomic engineering of transgenic crop plants prior to commercialization.
Expression of three pollen-specific promoters in transgenic rice plants. Crop biotechnology has the potential to improve the productivity of U.S. agriculture, but more tools are needed to precisely control gene expression in grass species like rice and wheat. Few well-characterized organ-specific promoters are available, particularly for expression in the non-seed organs of cereal grain crops. In 2011, ARS research scientists in the Crop Improvement/Utilization Research Unit in Albany, CA, completed characterization of three rice promoters that are active only in the mature pollen of transgenic rice plants. The promoters have slightly different cell specificities: one is active only in sperm cells, while the other two are active in pollen vegetative cells. These promoters will be useful tools for expressing genes that can modify metabolism in the pollen of rice and potentially other crop plants for control of male fertility and pollen-mediated gene flow.
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.
Wang, Y., Yau, Y., Perkins-Balding, D., Thomson, J.G. 2010. Recombinase Technology: Applications and Possibilities. Plant Cell Reports. 30(3):267-285.
Moon, H.S., Abercrombie, L.L., Eda, S., Blanvillain, R., Thomson, J.G., Ow, D.W., Stewart, N.C. 2011. Pollen-specific transgene excision in tobacco using the codon optimized serine resolvase CinHo-RS2 site-specific recombination system. Plant Molecular Biology. 75(6):621-631.