Location: Crop Improvement and Genetics Research2015 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Progress was made toward all objectives in Fiscal Year 2015. Towards meeting Subobjective 1a, a library of DNA sequences encoding recombinases was created for the production of mutants that could show enhanced enzymatic activity. The library employs a method that shuffles segments of genes from related family members to produce hybrid genes. Such a library was created from the small serine recombinase family that includes CinH, ParA, Tn1721 and Tn5053, each of which have been demonstrated to be active in eukaryotes. The vector for assaying activity of the hybrid enzymes is currently under construction and will be complete by the end of the calendar year. Identification of mutant recombinases that function in the bacterial assay will begin soon after. To meet Subobjective 1b, transgenic "Founder" lines of Camelina, tobacco and Arabidopsis containing target sites for Recombinase-Mediated Cassette Exchange (RMCE) have been generated and shown to contain one or two copies of the genomic target. These lines have been re-transformed with exchange vectors containing expression cassettes and recognition sites for site specific recombinases. Site-specific integration occurs in 30% of the Arabidopsis and tobacco lines, but the Camelina lines have proven recalcitrant to re-transformation. Investigation continues to determine whether site-specific excision (and thus completed exchange) has occurred in the re-transformed Arabidopsis and tobacco lines. Towards meeting Subobjective 2a, research investigating the function of four new candidate rice promoters with floral or reproductive expression and three novel root-specific promoters began. These previously uncharacterized organ-specific promoters were introduced into rice, Brachypodium, and other plants. Several of the promoters exhibit expression in the expected tissues in the regenerated rice transgenic plants. The characterization of three wheat promoters from genes that exhibit low basal levels of expression in healthy plants, but are induced by environmental or disease stresses, has continued. Transgenic wheat and Brachypodium plants carrying these wheat promoters have been generated and their activities are being evaluated and documented. Towards meeting Subobjective 2b, research developing an approach to identify native sequences that function as insulators of gene expression also continued. Several replicate validation experiments have been performed to confirm that the screening assay as designed can reliably differentiate functional insulating sequences from other non-insulating sequences. The construction of a library of size-fractionated rice sequences cloned into the insulator test vector is underway. This library will be used in the screening assay to identify sequences that function as transcription insulators in plants. In collaborative research, a biocontainment system for switchgrass was developed with an industrial partner. Since switchgrass is an important biofuel crop and is a wind-pollinated outcrossing species, a method of genetic containment for biotechnology traits was investigated. The use of pollen-specific promoters to control the expression of the Bxb1 recombinase was demonstrated to be a successful strategy to remove transgenes from switchgrass pollen. Although the containment system requires further optimization to achieve commercial levels of gene containment, this research establishes that reproductive expression of a site-specific recombinase in switchgrass can be used to excise target sequences preventing their inheritance in the next generation. In work funded by a National Institute of Food and Agriculture (NIFA) grant entitled “Improved Recombinase Technology for Targeted Marker Free Integration and Found Line Production for Risk Assessment” (2030-21000-020-01R), "Founder" lines of yeast containing integrated Bxb1 and CinH recombinase recognition site targets along with paired exchange (pEXCH) vectors for Recombinase Mediated Cassette Exchange (RMCE) were used to perform sequential genomic targeted integration of four separate genes. Both targeted integration and excision were highly efficient (68-100% RMCE targeting depending on the systems tested), but the excision reaction was difficult to control because the inducible promoter system used was “leaky”. The promoter will be modified to make prevent gene expression in the absence of the inducer. A highly efficient RMCE system for metabolic engineering of yeast will be a necessary component of future biofuel research. The consistency of transgene-encoded protein expression levels in yeast generated from this study is being investigated. An invention disclosure for this research was filed in a previous year. In work funded by a Citrus Research Board (CRB) grant entitled “Utilization of founder lines for improved Citrus biotechnology via RMCE” (2030-21000-020-11T), 335 different transgenic Founder line plants from variety ‘Carrizo’, and 15 from ‘Hamlin’ have been obtained. Southern blot analysis showed that 65 ‘Carrizo’ lines and 9 ‘Hamlin’ lines contained a single-copy RCME target site. Twenty-eight Founder lines from ‘Carrizo’ and 9 from ‘Hamlin’ have been transformed with the pEXCH vector. Double-transformed plants have been regenerated and analysis of site-specific targeted integration is underway. Discussions with CRB about intellectual property (IP) protection for the Founder lines is in progress. In research funded by a second CRB grant entitled “The Development of Novel Blood and Cara Cara like Citrus Varieties” (2030-21000-020-06T), five new promoters active in fruit were isolated from the citrus genome and four other candidates for fruit-specific promoters were isolated from other plant genomes. The tissue specificity of these promoters was evaluated in transgenic tomato and tobacco plants. Four of the five citrus and two promoters from the other species show promise for confining gene expression to fruit tissues. The remaining three promoters are either constitutive or seed-specific in expression pattern. IP protection of the fruit-specific promoters is under discussion with CRB. In research funded by the United Soybean Board entitled “Utilization of Founder Lines for Improved Soybean Biotechnology via RMCE” (2030-21000-020-07R), the initial three Founder lines in cultivar ‘Bert’ and the one line in ‘Magellian’ were deep sequenced to determine with better resolution the structure of the target transgene sites. These lines had been previously shown to be single copy by Southern blot analyses. Unexpectedly, the DNA sequencing showed that some of the lines contained "extra" pieces of the initial targeting vector. This will complicate RMCE testing of these Founder lines. Nevertheless, two of the four lines have been transformed with the pEXCH vector. Plant regeneration is underway. DNA from re-transformants will be analyzed to determine whether or not RCME (targeted integration and excision) has occurred. In U.S. Department of Energy funded research entitled, “Expanding the Breeder’s Toolbox” (2030-21000-020-12R), a strategy for reducing transgene flow to neighboring plants is being developed. Plant transformation vectors utilizing several of the pollen-specific promoters previously developed by the project have been shown to effectively ablate transgenic pollen in Brachypodium sylvaticum, a model perennial grass. These constructs are now being transformed into switchgrass. In addition, research on a strategy to ablate all floral development in hybrid transgenic plants has been pursued. Transgenic plants have been generated and are being crossed to make hybrids in which the floral ablation transgenes are activated. The next step is to evaluate how effectively reproductive development is altered in the hybrids.
Somleva, M.N., Chang, A.X., Ryan, K.P., Thilmony, R.L., Peoples, O., Snell, K.D., Thomson, J.G. 2014. Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase. BioMed Central (BMC)Biotechnology. 14:79. doi: 10.1186/1472-6750-14-79.
Thomson, J.G., Blechl, A.E. 2015. Recombinase technology for precise genome engineering. In: Zhang, F., Puchta, H., Thomson, J.G., editors. Advances in new technology for targeted modification of plant genomes. New York, NY: Springer. p. 113-144.
Shao, M., Michno, J., Blechl, A.E., Thomson, J.G. 2015. A bacterial gene codA encoding cytosine deaminase is an effective conditional negative selectable marker in Glycine max. BioMed Central (BMC)Biotechnology. doi: 10.1007/s00299-015-1818-5.
Thomson, J.G., Stover, E.W., Peixoto De Olivei, M.L. 2015. The codA gene as a negative selection marker in Citrus. BioMed Central (BMC)Biotechnology. 4:264.