Abstract
Introns, especially the first intron in the 5’ untranslated region (5’UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.
Highlights
Eukaryotic elongation factor 1A genes, encoding one of the most abundant soluble proteins in plant cells [1], are constitutively expressed in most tissues, with high expression in rapidly growing tissues, such as shoot and root meristems and developing gametophytes [2]
With the constitutively-expressed promoters and regulatory regions selected for this study, gene expression intensities measured using transient expression were largely confirmed in stably-transformed roots
Our results suggested that a 222 bp intronic sequence was an important contributor to intron-mediated enhancement (IME) from the GmScreamM8 leader intron (Figs 4–6)
Summary
Eukaryotic elongation factor 1A (eEF1A) genes, encoding one of the most abundant soluble proteins in plant cells [1], are constitutively expressed in most tissues, with high expression in rapidly growing tissues, such as shoot and root meristems and developing gametophytes [2]. EEF1A proteins are multifunctional proteins and interact with major cytoskeletal proteins to organize the cytoskeleton structure by binding to tubulin, actin or other microtubule associated proteins [4]. Eukaryotic eEF1A genes exhibit a highly conserved structure, each with a relatively large intron in its 5’ untranslated region (5’ UTR). An Arabidopsis eEF1A promoter and its 5’ UTR intron was used in the herbicide resistance gene in commercial RoundupReady soybean plants [10], but the specific reason for use of those sequences has not been reported
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