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2020
Corte, L. Erpen Dalla; Mendes, B. M. J.; Filho, F. A. A. Mourão; Grosser, J. W.; Dutt, M.
Functional characterization of full-length and 5′ deletion fragments of Citrus sinensis-derived constitutive promoters in Nicotiana benthamiana Journal Article
In: In Vitro Cellular and Developmental Biology - Plant, vol. 56, iss. 3, pp. 280-289, 2020, ISSN: 14752689.
Abstract | Links | BibTeX | Tags: Cis-regulatory elements, Gene expression, genetic transformation, sweet orange
@article{Corte2020,
title = {Functional characterization of full-length and 5′ deletion fragments of Citrus sinensis-derived constitutive promoters in Nicotiana benthamiana},
author = {L. Erpen Dalla Corte and B. M. J. Mendes and F. A. A. Mourão Filho and J. W. Grosser and M. Dutt},
url = {https://link.springer.com/article/10.1007/s11627-019-10044-0},
doi = {10.1007/S11627-019-10044-0/FIGURES/7},
issn = {14752689},
year = {2020},
date = {2020-01-01},
journal = {In Vitro Cellular and Developmental Biology - Plant},
volume = {56},
issue = {3},
pages = {280-289},
publisher = {Springer},
abstract = {Full-length and 5′ deletion fragments of three constitutively expressed gene promoters identified from the Citrus sinensis L. genome (cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1)) were evaluated for their ability to express the uidA gene in leaf, stem, and root tissues of transgenic N. benthamiana plants. According to the fluorometric GUS assays, the CsGAPC2 promoter activity was significantly reduced in the leaves when the sequence between position − 1090 and − 497 was removed, while the activity remained unchanged in the roots. The CsCYP promoter activity was not affected by the different deletions, and even the smallest evaluated fragment was sufficient to maintain the expression of the uidA gene in N. benthamiana leaves and roots. Truncated promoter fragments of CsEF1 conferred higher GUS activity in leaves compared with the full-length promoter, while GUS activity was not affected in the roots. The removal of the intron in the 5′ UTR of the CsEF1 promoter reduced gene expression in the roots, although it did not cause any reduction in the gene expression level in the leaves. These results indicate that any of the three deletions of the CsCYP promoter can be utilized for efficient gene expression. In addition, the full-length CsGAPC2 promoter and two of the truncated CsEF1 promoter fragments were sufficient for efficient uidA gene expression.},
keywords = {Cis-regulatory elements, Gene expression, genetic transformation, sweet orange},
pubstate = {published},
tppubtype = {article}
}
Full-length and 5′ deletion fragments of three constitutively expressed gene promoters identified from the Citrus sinensis L. genome (cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1)) were evaluated for their ability to express the uidA gene in leaf, stem, and root tissues of transgenic N. benthamiana plants. According to the fluorometric GUS assays, the CsGAPC2 promoter activity was significantly reduced in the leaves when the sequence between position − 1090 and − 497 was removed, while the activity remained unchanged in the roots. The CsCYP promoter activity was not affected by the different deletions, and even the smallest evaluated fragment was sufficient to maintain the expression of the uidA gene in N. benthamiana leaves and roots. Truncated promoter fragments of CsEF1 conferred higher GUS activity in leaves compared with the full-length promoter, while GUS activity was not affected in the roots. The removal of the intron in the 5′ UTR of the CsEF1 promoter reduced gene expression in the roots, although it did not cause any reduction in the gene expression level in the leaves. These results indicate that any of the three deletions of the CsCYP promoter can be utilized for efficient gene expression. In addition, the full-length CsGAPC2 promoter and two of the truncated CsEF1 promoter fragments were sufficient for efficient uidA gene expression.
2018
Erpen, L.; Tavano, E. C. R.; Harakava, R.; Dutt, M.; Grosser, J. W.; Piedade, S. M. S.; Mendes, B. M. J.; Filho, F. A. A. Mourão
Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters Journal Article
In: Plant Cell Reports, vol. 37, iss. 8, pp. 1113-1125, 2018, ISSN: 07217714.
Abstract | Links | BibTeX | Tags: Cis-regulatory elements, Cultivar improvement, Gene expression, sweet orange
@article{Erpen2018,
title = {Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters},
author = {L. Erpen and E. C. R. Tavano and R. Harakava and M. Dutt and J. W. Grosser and S. M. S. Piedade and B. M. J. Mendes and F. A. A. Mourão Filho},
url = {https://link.springer.com/article/10.1007/s00299-018-2298-1},
doi = {10.1007/S00299-018-2298-1/METRICS},
issn = {07217714},
year = {2018},
date = {2018-01-01},
journal = {Plant Cell Reports},
volume = {37},
issue = {8},
pages = {1113-1125},
publisher = {Springer Verlag},
abstract = {Key message: Regulatory sequences from the citrus constitutive genes cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1) were isolated, fused to the uidA gene, and qualitatively and quantitatively evaluated in transgenic sweet orange plants. Abstract: The 5′ upstream region of a gene (the promoter) is the most important component for the initiation and regulation of gene transcription of both native genes and transgenes in plants. The isolation and characterization of gene regulatory sequences are essential to the development of intragenic or cisgenic genetic manipulation strategies, which imply the use of genetic material from the same species or from closely related species. We describe herein the isolation and evaluation of the promoter sequence from three constitutively expressed citrus genes: cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1). The functionality of the promoters was confirmed by a histochemical GUS assay in leaves, stems, and roots of stably transformed citrus plants expressing the promoter-uidA construct. Lower uidA mRNA levels were detected when the transgene was under the control of citrus promoters as compared to the expression under the control of the CaMV35S promoter. The association of the uidA gene with the citrus-derived promoters resulted in mRNA levels of up to 60–41.8% of the value obtained with the construct containing CaMV35S driving the uidA gene. Moreover, a lower inter-individual variability in transgene expression was observed amongst the different transgenic lines, where gene constructs containing citrus-derived promoters were used. In silico analysis of the citrus-derived promoter sequences revealed that their activity may be controlled by several putative cis-regulatory elements. These citrus promoters will expand the availability of regulatory sequences for driving gene expression in citrus gene-modification programs.},
keywords = {Cis-regulatory elements, Cultivar improvement, Gene expression, sweet orange},
pubstate = {published},
tppubtype = {article}
}
Key message: Regulatory sequences from the citrus constitutive genes cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1) were isolated, fused to the uidA gene, and qualitatively and quantitatively evaluated in transgenic sweet orange plants. Abstract: The 5′ upstream region of a gene (the promoter) is the most important component for the initiation and regulation of gene transcription of both native genes and transgenes in plants. The isolation and characterization of gene regulatory sequences are essential to the development of intragenic or cisgenic genetic manipulation strategies, which imply the use of genetic material from the same species or from closely related species. We describe herein the isolation and evaluation of the promoter sequence from three constitutively expressed citrus genes: cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1). The functionality of the promoters was confirmed by a histochemical GUS assay in leaves, stems, and roots of stably transformed citrus plants expressing the promoter-uidA construct. Lower uidA mRNA levels were detected when the transgene was under the control of citrus promoters as compared to the expression under the control of the CaMV35S promoter. The association of the uidA gene with the citrus-derived promoters resulted in mRNA levels of up to 60–41.8% of the value obtained with the construct containing CaMV35S driving the uidA gene. Moreover, a lower inter-individual variability in transgene expression was observed amongst the different transgenic lines, where gene constructs containing citrus-derived promoters were used. In silico analysis of the citrus-derived promoter sequences revealed that their activity may be controlled by several putative cis-regulatory elements. These citrus promoters will expand the availability of regulatory sequences for driving gene expression in citrus gene-modification programs.