Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues

Abstract

BackgroundCotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization.ResultsNucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2.ConclusionsuceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.

Isolation and Characterization of Three New Promoters from Gossypium hirsutum that Show High Activity in Reproductive Tissues

“…The expression pattern of the GhPME-like1 , GhβGal-like1 and GhPL-like1 genes in cotton agrees in principal with the regulatory pattern of their promoters because lower activity was observed in vegetative tissues and much higher activity was observed in flower buds, inflorescences and open flowers. The pGhPME-like1 , pGhβGal-like1 and pGhPL-like1 cotton promoters were compared with the uceA1.7 cotton promoter previously described by Viana et al (2011), and they showed significantly higher activity in Arabidopsis reproductive organs (data not shown). Histochemical assays of 6-mm cotton flower buds that had been bombarded with the tissue-specific promoters p300_GhPME-like1 (a), p760_GhPME-like1 (b), p1618_GhPME-like1 (c), pGhβGal-like1 (d) or pGhPl-like1 (e).…”

Identification and functional characterization of the MdHB-1 gene promoter sequence from Malus×domestica

“…and the exact position of the structural domain needs to be verified by additional experiments. Previous studies reported that 5´-UTR could act as an enhancer in the gene expression process (Viana et al 2011;Kamo et al 2012;Kanoria and Burma 2012;Agarwal et al 2014). In absence of 5´-UTR region, MH (-1057 to -266) had a weaker GUS activity than MH10, which implies that 5´-UTR may regulate the MdHB-1 gene expression.…”

Alterations in the 5′ untranslated region of the 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene influence EPSPS overexpression in glyphosate‐resistant Eleusine indica

“…It confers high transcription levels without requiring other upstream cis-elements, except for the TATA box. 33 Studies on the promoter function of the amorpha-4,11-diene synthase (ADS) gene in Artemisia annua 34 and the high-molecular-weight glutenin subunit (HMW-GS) gene in Triticum aestivum 35 demonstrated that the 5 ′ UTR Py-rich stretch elements were important factors in the enhancement of the expression of the ADS and HMW-GS genes. The mutation length of the 5 ′ UTR Py-rich stretch motif influences promoter activity of the tryptophan decarboxylase (tdc) gene in Catharanthus roseus.…”