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ISOLATION AND CHARACTERIZATION OF SOYBEAN PROMOTERS

Hernandez-Garcia, Carlos M
http://rave.ohiolink.edu/etdc/view?acc_num=osu1366303238

Abstract Details

2013, Doctor of Philosophy, Ohio State University, Horticulture and Crop Science.
Promoters are DNA sequences located upstream of gene coding sequences. Since promoters largely control gene expression at the transcriptional level, promoter studies are crucial to improving our understanding of gene regulation and in developing improved transgenic crops. Based on their expression patterns, plant promoters are classified as constitutive, spatiotemporal and inducible. Large-scale prediction of promoter sequences and their contributing cis-acting elements is now feasible because of recent advances in transcriptomic technologies, improvements in computer modeling and the availability of increasing numbers of genome sequences for different plant species. These approaches reveal a large number of cis-elements that need further validation utilizing gene expression systems. A major effort in promoter evaluation and promoter element identification has been the development and improvement of expression tools for rapid, efficient, predictable, and high-throughput analysis of promoter constructs. The use of synthetic promoters specifically designed for characterization of cis-elements could also facilitate the investigation of promoter functionality, and lead to the development of transgenic plants with enhanced gene expression and more elaborated phenotypes. In this research, I focus on the constitutive Ubiquitin and the wound responsive Ethylene Response Factor (ERF) gene promoters. I initially developed and optimized two gene expression validation tools for rapid characterization of numerous promoter constructs utilizing a lima bean cotyledonary-transient expression system and stably-transformed soybean hairy roots. These validation tools were central to study the gene expression mediated by 20 novel soybean promoters isolated here from the Gmubi and GmERF gene families. The Gmubi promoters mediated strong GFP expression in both validation tools and are good candidate promoters to drive strong constitutive expression in transgenic plants. In transgenic soybeans, the Gmubi promoter from the polyubiquitin Gmubi3 gene mediated exceptional high levels of GFP in different organs and tissues of transgenic soybeans. For inducible promoters, I studied the gene expression patterns driven by the soybean GmERF3 promoter and its cis-acting elements in transgenic tobacco and soybean. The GmERF3 promoter mediated delayed induction of GFP after mechanical wounding in soybean leaves. A combination of in silico and functional analyses, including deletion analysis and synthetic promoters led to the identification of a novel cis-element namely LWI (Late Wound Inducible) element. In transgenic tobacco, the LWI element resembled similar wound-induced expression patterns than the full-length GmERF3 promoter. In conclusion, this study expands the toolbox of strong constitutive promoters and wound-inducible promoters and cis-acting elements for potential use in basic and applied research.
John Finer (Advisor)
Michelle Jones (Committee Member)
Eric Stockinger (Committee Member)
Anne Dorrance (Committee Member)
Bioinformatics; Cellular Biology; Molecular Biology

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Citations

  • Hernandez-Garcia, C. M. (2013). ISOLATION AND CHARACTERIZATION OF SOYBEAN PROMOTERS [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366303238

    APA Style (7th edition)

  • Hernandez-Garcia, Carlos. ISOLATION AND CHARACTERIZATION OF SOYBEAN PROMOTERS. 2013. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1366303238.

    MLA Style (8th edition)

  • Hernandez-Garcia, Carlos. "ISOLATION AND CHARACTERIZATION OF SOYBEAN PROMOTERS." Doctoral dissertation, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366303238

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.