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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

2014 Annual Report

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.

Progress Report
Progress on the first objective includes the completion of characterization of the activities of several tissue-specific promoters (conferring leaf-, root-, pollen- and endosperm-specific expression specificities) in transgenic rice, wheat and/or Brachypodium distachyon plants. The claims of a patent application (12/890,974) for the LP2 promoter that confers light-inducible gene expression in green tissues were recently allowed by the U.S. Patent and Trademark Office. Research investigating the function of four new candidate promoters with floral or reproductive expression has begun. These novel organ-specific promoters have been introduced into rice, Brachypodium, switchgrass and other 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 promoters have been generated and their activities are being evaluated and documented. In support of the second objective, random mutations were generated by Polymerase Chain Reaction (PCR) in the genes of site-specific recombinases Bxb1, U153 and TP901. The variant enzymes were screened for excisionase activity in a bacterial assay and sequence analysis determined the exact nature of the mutations in the Bxb1 and U153 genes. Mutant recombinases with improved activity are being identified. Concurrently, “target” vectors (pCTAG) were generated that contain the recombinase recognition sites required for recombinase mediated cassette exchange (RMCE) flanking a positive-negative selectable marker system (codA-nptII). Further, the initial “exchange” vector (pEXCH) was constructed to test four pairs of uni-directional recombinases. It was designed so that an incoming gene is integrated at the target site and the selection marker genes (codA-nptII) are excised in a two-step sequential process. Because pEXCH is a large plasmid intended for use in various plant species, the construction is being redesigned for easy cloning purposes. In work funded by a National Institute of Food and Agriculture (NIFA) grant (5325-21000-020-01R), “Founder Lines” of yeast containing integrated target vectors were obtained for each of four recombinase enzyme pairs. The pEXCH vectors to test RCME for each pair of recombinases were constructed and transformed into the Founder Line that contained their target recognition sites. The frequency of RMCE was measured and compared among the different recombinase pairs. The Bxb1/CinH pair was the most efficient for RCME, followed closely by the phiC3/CinH pair. The other two pairs, Bxb1/ParA and phiC31/ParA, were significantly less efficient. These data will be used to speed RMCE testing in the plant Founder Lines. In work funded by a Citrus Research Board (CRB) grant (5325-21000-020-04T), 335 different transgenic Founder Line plants from variety ‘Carrizo’, and 15 from ‘Hamlin’ were obtained. The insertion of the target construct pCTAG was confirmed in each by PCR. Southern blot analysis showed that 65 ‘Carrizo’ lines and 9 ‘Hamlin’ lines contained a single pCTAG insertion. These lines are being used for RMCE testing with the pEXCH vector that was constructed with funding from a second CRB grant (0000051713). Twenty-eight Founder Lines from ‘Carrizo’ and 9 from ‘Hamlin’ have been transformed with the pEXCH vector and plant regeneration is underway. The transformants will be subjected to molecular analyses to determine whether or not RCME has occurred. In research funded by a third CRB grant (5325-21000-020-06T), five new promoters active in fruit were isolated from the citrus genome. Four other candidate fruit-specific promoters were isolated from a variety of other plant genomes. The tissue specificity of these promoters will be evaluated in transgenic plants. In research funded by the United Soybean Board (5325-21000-020-07R), the initial pEXCH vector was constructed and transformed into three Founder Lines of cultivar ‘Bert’. Plant regeneration is underway and transformants will be subjected to molecular analyses to determine whether or not RCME has occurred. In U.S. Department of Energy funded research (5325-21000-020-05I), a strategy for reducing pollen-mediated transgene flow is being developed. Plant transformation vectors that utilize several of the pollen-specific promoters developed by the project have been constructed and transformed into Brachypodium plants. Initial results suggest that transgenic pollen is being ablated in the transformed plants. A detailed examination is underway to evaluate the viability of the transgenic pollen from these plants and the transmission of transgenes via the pollen to non-transgenic plants in crosses.

1. A new molecular tool for making precise changes in plant chloroplast DNA. New methods and tools are needed to facilitate making precise changes in plant DNA. Towards that goal, ARS scientists in Albany, California, have been developing and testing several recombinase enzymes from single celled organisms for their ability to function efficiently in plant cells. In 2014, they showed that the Bxb1 recombinase can precisely remove DNA from the tobacco plastid genome and that the modified DNA is inherited by progeny plants. Because the Bxb1 enzyme does not add or delete nucleotides outside its recognition domain, the integrity of the surrounding chloroplast genes is maintained. These results demonstrate that Bxb1 is a unique molecular tool that can be used to remove unwanted antibiotic or herbicide resistance genes after genetic engineering of chloroplast DNA. The outcome is reuse of these selection markers for further rounds of transformation or release of the plants without resistance genes into commercial production.

Review Publications
Shao, M., Kumar, S., Thomson, J.G. 2014. Precise excision of plastid DNA by the large serine recombinase Bxb1. Plant Biotechnology Journal. 12:322-329 DOI: 10.1111/pbi.12139.
Thilmony, R.L., Guttman, M.E., Lin, J.W., Blechl, A.E. 2014. The wheat HMW-glutenin 1Dy10 gene promoter controls endosperm expression in Brachypodium distachyon. GM Crops. 5:36-43.
Wang, Y., Thilmony, R.L., Gu, Y.Q. 2014. Net Venn - An integrated network analysis web platform for gene lists. Nucleic Acids Research. DOI: 10.1093/narlgku331.