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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Tools for Improved Crop Biotechnology

Location: Crop Improvement & Utilization Research

Project Number: 5325-21000-020-00
Project Type: Appropriated

Start Date: Jul 23, 2013
End Date: Jul 22, 2018

The overall goal of the project is to identify DNA elements that support effective strategies for stacking multiple traits within a single locus, removal of unwanted DNA sequences, and predictable expression of each transgene within that locus. These molecular tools will enable improved and precise engineering of complex, multi-gene traits in crop plants. Site-specific recombination systems and gene expression control elements with proven utility will be made available to researchers in the public and private sectors. Objective 1: Develop and deploy in crop plants site-specific recombinase-based systems for (1) targeted transgene integration and gene stacking, and (2) marker gene removal to prevent gene flow to non-genetically engineered crops. Subobjective 1a: Enhance site specific recombination systems for precise integration and excision in crop plant cells. Subobjective 1b: Use Dual RMCE to produce Foundation Lines that will allow transgene stacking via reiterative targeted integration and marker gene removal. Objective 2: Identify and demonstrate the utility of crop-derived gene expression control elements (promoters/enhancers/terminators/insulators) that facilitate trait development in crop plants. Subobjective 2a: Isolate and characterize novel promoters. Subobjective 2b: Isolate and characterize novel transcription terminators.

Random mutagenesis will be used to generate site-specific recombinase variants that will be screened for improved integrase and excisionase activities in a recombinase activity assay. Versions with improved catalytic activities in bacterial cells will be tested in plant cells. Mutated recombinases with improved activity will be codon optimized and tested in transgenic plants. In parallel, “target” transgenic plants will be generated by Agrobacterium–mediated transformation of Camelina. “Exchange” T-DNA vectors will be constructed to test four pairs of uni-directional recombinases, and designed so that an incoming gene is integrated at the target site and the selection marker gene is excised in a two-step sequential process. The “exchange” vectors will be transformed into the “target” Camelina transgenic plants. Negative selection will be used to screen for transformants in which the incoming DNA has replaced the original transgenic locus (Recombinase-Mediated Cassette Exchange or RMCE). The resultant transgene structure will be molecularly characterized to demonstrate that cassette exchange and selection marker gene removal have occurred. The efficiencies of different combinations of the unidirectional recombinases in performing RMCE will be compared. Candidate promoters with new cell-type/organ or stress-responsive specificities will be identified from crop plants using gene expression analyses. Emphasis will be on selecting candidates that have potentially useful expression patterns, but are not expressed in the grain. The candidate promoters will be fused to a reporter gene and transformed into rice, wheat, Brachypodium distachyon and/or other plants using Agrobacterium tumefaciens or biolistic transformation methods. Novel transcription terminator sequences will also be isolated from crop plants and fused to a reporter gene. The functionality of these promoter and terminator testing constructs will be examined in transient expression assays and stably transformed transgenic plants. Reporter gene expression levels will be quantitatively measured in major organs and compared to identify the sequences that provide the highest levels of transgene products while preserving promoter expression specificity. Additionally, a screen to identify “insulator” sequences that protect the expression of transgenes from undesirable interactions with nearby enhancers will be performed using a construct containing two copies of the highly active 35S enhancer. A library of crop genomic sequences will be tested for insulation activity using a transient expression assay. Selected candidate insulator sequences will also be tested in stably transformed transgenic plants. The functionality of the candidate insulator sequences will be validated if their insertion between the double 35S enhancer and a test promoter preserves the native specificity of the test promoter.

Last Modified: 4/23/2014
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