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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Research Project #425198

Research Project: Molecular Tools for Improved Crop Biotechnology

Location: Crop Improvement and Genetics Research

2017 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 both of the objectives during this year. In research related to Subobjective 1a, a novel gene stacking system was developed that enables the sequential, modular assembly of plant transformation constructs. Recombinase-mediated gene stacking is performed within bacteria by a process of sequential rounds of bacterial transformation with a donor plasmid and alternating antibiotic selection. To demonstrate the functionality of the strategy, five genes have been sequentially stacked into a novel Agrobacterium strain and this strain has been successfully used to generate transgenic Arabidopsis and potato plants. Subobjective 1b: Transgenic lines of tobacco and Arabidopsis containing target sites for Recombinase-Mediated Cassette Exchange (RMCE) have been generated and re-transformed with exchange vectors containing various expression cassettes for site-specific recombination. The system has been modified to increase the amount of ‘cargo’ DNA that can be carried and the levels of effective recombinase enzyme being expressed. Rates of integration now range from nineteen to seventy-eight percent in the tobacco lines tested. Site-specific integration has been validated by use of Polymerase Chain Reaction (PCR) to amplify the new DNA junctions. Subobjective 2a: Research investigating the function of four novel root-specific promoters was completed. These promoters confer expression within specific cell types of the roots of transgenic rice and tobacco plants. In addition, two candidate promoters that confer expression in vegetative tissues, but not floral reproductive tissues were isolated. The characterization of these promoters in transgenic plants is in progress. Subobjective 2c: Several candidate rice sequences that function in a rapid screening assay as insulators to shield genes from the effects of neighboring DNA sequences were selected for further characterization. These insulator sequences were moved into a plant transformation construct and introduced into tobacco plants. One of the candidate insulators exhibited enhancer-blocking activity in the transgenic tobacco and is available for further characterization and potential use in insulating transgene expression in crop plants. The novel use of the droplet digital PCR technique was developed to count the number of transgenes present in genetically engineered rice, wheat, tomato, citrus, maize, Arabidopsis and Brachypodium plants. This technique enables the more efficient characterization of transgenic plants, compared to previously used methods. Efforts to expand the use of this technology in switchgrass, barley and other crop species are underway. In research funded by grants, “Development of Mature Budwood Transformation Technology” (2030-21000-020-13T) and “The Development of Novel Blood and Cara Cara Like Citrus Varieties” (2030-21000-020-06T) from the Citrus Research Board, improvements were made in the methods to obtain and identify transgenic citrus plants. Media and culture conditions were optimized to use mature budwood tissue as the target explants for transformation of some varieties. Spectinomycin showed promise as an alternative to kanamycin as a selection agent. Four anti-apoptosis (anti-death) and seven early embryogenesis genes were cloned and are currently being evaluated for their ability to enhance transformation and regeneration, respectively. In research funded by the National Institute of Food and Agriculture (NIFA) in collaboration with scientists at the New Mexico Consortium, “Design and Delivery of Therapeutic Proteins for HLB Protection” (2030-21000-020-20R), seven genes (CSM1, mThionin, NPR1, PDR1.2, hrf1, SCAMPPS and PME) with the potential to combat citrus greening disease have been obtained and incorporated into vectors for citrus transformation and expression. They are being introduced into 'Carrizo' and 'Mexican Lime' varieties.

4. Accomplishments
1. Regulatory genes to improve the nutritional properties of citrus and other fruit. Anthocyanins are typically red or purple plant pigments that confer color and anti-oxidant properties in fruits and flowers. To increase the levels of these beneficial nutrients in foods, ARS scientists in Albany, California, isolated three new regulatory plum genes that encode protein switches with the potential to activate the production of anthocyanins. They also synthesized a fourth regulatory gene, modeling it on the equivalent gene from citrus trees. When these genes were introduced into tobacco plants, the citrus gene and two of the plum genes elicited the accumulation of anthocyanin pigments in various tissues, but with distinctly different patterns and intensities. These novel genes can now be used to activate the desirable production of anthocyanins in fruits, enhancing both their attractiveness and nutritional quality for consumers. These genes could also be used as simple and non-destructive visual markers for plant transformation.

2. New method to measure transgene copy number in genetically engineered crop plants. Detailed characterization of the DNA insertion sites in transgenic crop plants is required for basic research and for petitions to U.S. Federal regulatory agencies for de-regulation prior to commercialization. The genome sizes of crop plants vary over a 200-fold range and this makes accurate determination of the numbers of gene copies added to them by genetic transformation very difficult. In the past, these determinations have relied on cumbersome DNA blot or quantitative Polymerase Chain Reaction (PCR) methods. This year, ARS scientists in Albany, California, and Fort Pierce, Florida, adapted the drop digital PCR method to quantify the number of transgene copies in the genomes of genetically engineered rice, tomato, citrus, potato, maize and bread wheat plants. The technique is so sensitive that it can distinguish one from two copies, even among the 180 trillion base pairs that comprise the genome of hexaploid bread wheat. The new method gives biotechnologists a more robust and sensitive way to measure the copy numbers of added genes in transgenic crop plants and will greatly facilitate the choice of which transformation events merit more detailed study.

Review Publications
Dasgupta, K., Thilmony, R.L., Stover, E.W., Oliveira, M.L., Thomson, J.G. 2017. Novel R2R3-MYB transcription factors from Prunus americana regulate differential patterns of anthocyanin accumulation in tobacco and citrus. GM Crops & Food. 8:85-105.
Shao, M., Blechl, A.E., Thomson, J.G. 2017. Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA. Plant Biotechnology Journal. doi: 10.1111/pbi.12740.
Collier, R.A., Dasgupta, K., Xing, Y., Hernandez, B., Shao, M., Rohozinski, D., Kovak, E., Lin, J.W., De Oliveira, M., Stover, E.W., Mc Cue, K.F., Harmon, F.G., Blechl, A.E., Thomson, J.G., Thilmony, R.L. 2017. Accurate measurement of transgene copy number in crop plants using droplet digital PCR. Plant Journal. 90:1014-1025.
Valdes, J., Collier, R.A., Wang, Y., Huo, N., Gu, Y.Q., Thilmony, R.L., Thomson, J.G. 2002. Draft genome sequence of Agrobacterium rhizogenes strain NCPPB2659. Genome Announcements. 4:1-2.