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Dissection of GmScream Promoters that Regulate Highly Expressing Soybean (Glycine max Merr.) Genes

Zhang, Ning
http://orcid.org/0000-0003-2775-1755
http://rave.ohiolink.edu/etdc/view?acc_num=osu1469052454

Abstract Details

2016, Doctor of Philosophy, Ohio State University, Horticulture and Crop Science.
Promoters and their contributing cis-elements are among the most important regulatory components for transcriptional regulation of gene expression. Studies on plant promoters and their cis-regulatory elements hold great promise for both basic research and agricultural biotechnology. Here, a group of twenty “GmScream” promoters, which regulate highly expressed soybean genes, were identified and characterized using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Out of these promoters, one half showed activity that was higher than or comparable to the widely-used Cauliflower Mosaic Virus 35S (CaMV35S) promoter. The strongest promoters, GmScreamM8 and GmScreamM4, drove high expression in most tissues of transgenic soybeans, with preferential high expression in young or rapidly growing tissues. The leader intron of GmScreamM8 significantly contributed to the observed high activity of the promoter through intron-mediated enhancement (IME). Element tetramers, placed upstream of the GmScreamM8 core promoter, contributed to very high activity, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Through the use of synthetic promoters and synthetic introns, we successfully identified a 222 bp intronic sequence that significantly contributed to intron-mediated enhancement. Promoter elements conferring high levels of promoter activity from several strong GmScream promoters were also identified in this study. To improve our understanding of how these short DNA sequences regulate gene expression, synthetic promoters consisting of a classical G-box core sequence (CACGTG) along with different flanking sequences were constructed and used to regulate a green fluorescent protein (gfp) gene. The proximal 2-bp flanking sequences of the G-box greatly affected G-box activity, while the distal 2-bp flanking nucleotides also influenced gene expression albeit at lower levels. Manipulation of the upstream 4-bp flanking sequence of a G-box in the proximal region of a native weak GmScream3 promoter significantly enhanced promoter activity. In addition, truncation analysis of the GmScreamM8 promoter revealed that a short DNA fragment located close to the transcriptional start site (TSS) was essential for increased gene expression. Using site-specific mutagenesis of targeted nucleotides in synthetic promoters, a novel GTAA element (GGTAAA/TTTA/CGTAA) was identified and functionally characterized as the regulatory element contributing to high gene expression in young and rapidly growing tissues including shoot meristems, root tips and proliferative embryogenic tissues in transgenic tobacco and soybean plants.
John Finer (Advisor)
Leah McHale (Committee Member)
Esther van der Knaap (Committee Member)
Feng Qu (Committee Member)
197 p.
Horticulture
GmScream promoters; intron-mediated enhancement; cis-regulatory elements; G-box; GTAA; GFP

Recommended Citations

Citations

  • Zhang, N. (2016). Dissection of GmScream Promoters that Regulate Highly Expressing Soybean (Glycine max Merr.) Genes [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469052454

    APA Style (7th edition)

  • Zhang, Ning. Dissection of GmScream Promoters that Regulate Highly Expressing Soybean (Glycine max Merr.) Genes. 2016. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1469052454.

    MLA Style (8th edition)

  • Zhang, Ning. "Dissection of GmScream Promoters that Regulate Highly Expressing Soybean (Glycine max Merr.) Genes." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469052454

    Chicago Manual of Style (17th edition)

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