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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Tools to Minimize Risk in Genetically Engineered Crops

Location: Crop Improvement & Utilization Research

2012 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:
Research characterizing several novel gene promoters activated in response to stress or pathogen attack continued. Several wheat genes that exhibit low basal levels of expression in healthy plants, but are induced by environmental or disease stress were identified. The upstream promoter sequences for 3 selected candidates, one responsive to both wounding and pathogen stress, and two others specifically responsive to disease stress, were isolated. Several plant transformation vectors including some that contain a BAR/cre fusion gene and a single lox site, were constructed by fusing these promoters to a reporter gene. Transgenic wheat and Brachypodium distachyon plants were produced with constructs containing one of these promoters. Characterization of reporter gene expression in the transgenic plants has begun and screening of their progeny is underway. Characterization of several rice organ-specific promoters has also continued. Research documenting the function of three novel rice promoters that are active only in the pollen of transgenic rice plants is nearly complete. Transformation and testing of these same promoters in transgenic Brachypodium and switchgrass has been initiated.

Research has also continued to develop site-specific recombination systems that are useful in plants. Functional Bxb1 recombinase was shown to be inherited in both transgenic wheat and Arabidopsis, where it precisely excised DNA flanked by its target sequences. To investigate their utility in other crop plants, constructs encoding the Bxb1, phiC31, CinH and ParA recombinases have been transformed into soybean, citrus, and the biofuel crop Camelina sativa. Development of a reliable transient assay system for direct comparison of the excision and/or integration efficiencies of the different recombinases in plant cells has been completed. The comparisons included the well-characterized Cre, Flp and R recombinases as well as the wild type and plant codon-optimized versions of our recombinases. Results from this system indicate that placement of the recombinase recognition sites within a construct can play a crucial role in their activity. Both enhancement and repression of site-specific recombination was observed, relative to the Cre recombinase which was used as an internal control, and guidelines for the placement of target sites were determined. With the goal of improving genomic targeting in crop plants, we have initiated development of an accelerated evolution system for increasing the integration activities of some of the recombinases.

1. Bxb1 recombinase catalyzes the reactions needed for precise plant genome engineering. Among the general public’s concerns about crop genetic engineering are.
1)the randomness of DNA integration into the chromosome(s) and.
2)the presence of antibiotic and herbicide resistance genes used to identify transformed plants, but not needed in the final plant. To improve the precision of plant biotechnology, scientists at the ARS in Albany, California, tested the functionality of the Bxb1 recombinase in model and crop plants. Bxb1 was shown to perform site-specific integration in tobacco. In Arabidopsis, wheat and switchgrass, the recombinase catalyzed the precise deletion of DNA situated between its two recognition sites. These experiments demonstrate the utility of the Bxb1 recombinase as a new tool for several aspects of plant genomic engineering including excision of marker genes, resolution of complex insertion structures, targeted DNA integration, and transgene stacking.

2. Identification of a rice sperm cell-specific promoter. Crop biotechnology has the potential to improve the productivity of U.S. agriculture, but 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. ARS scientists in Albany, California, identified a sperm cell-specific promoter (named OsGEX2) and characterized it in transgenic rice plants. The promoter reproducibly confers expression in the pollen, particularly the sperm cells, but exhibits low or undetectable expression in other parts of the plant including the leaves, the roots, other reproductive tissues, and the seeds. The OsGEX2 promoter will enable precise, localized expression of transgenes in the pollen of rice and other cereal grain crops and limit the potential for unintended impacts on the grain used for food and feed.

Review Publications
Cook, M.A., Thilmony, R.L. 2012. The OsGEX2 gene promoter confers pollen sperm cell-specific expression in transgenic rice. Planta. DOI: 10.1007/s11105-012-0429-3.

Thomson, J.G., Chan, R., Smith, J.D., Thilmony, R.L., Yau, Y., Wang, Y., Ow, D.W. 2012. The Bxb1 recombination system demonstrates heritable transmission of site-specific excision in Arabidopsis. BioMed Central (BMC)Biotechnology. 12:9 doi:10.1186/1472-6750-12-9.

Last Modified: 4/17/2014
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