<i>Agrobacterium tumefaciens</i>-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—<i>Euglena gracilis</i>
<i>Euglena gracilis</i> (<i>E. gracilis</i>) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of...
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MDPI AG
2021-06-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/12/6299 |
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| author | Ina Becker Binod Prasad Maria Ntefidou Viktor Daiker Peter Richter Michael Lebert |
| author_facet | Ina Becker Binod Prasad Maria Ntefidou Viktor Daiker Peter Richter Michael Lebert |
| author_sort | Ina Becker |
| collection | DOAJ |
| description | <i>Euglena gracilis</i> (<i>E. gracilis</i>) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of <i>E. gracilis</i> mediated by <i>Agrobacterium</i> (<i>A. tumefaciens</i>). <i>E. gracilis</i> was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. <i>A. tumefaciens</i>-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the <i>gus</i> gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the <i>gus</i> gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (<i>hptII</i>) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of <i>Euglena</i> with the <i>hptII-2A–gus</i> gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when <i>A. tumefaciens</i> LBA4404 (A<sub>600</sub> = 1.0) and <i>E. gracilis</i> (A<sub>750</sub> = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer <i>E. gracilis</i> for producing high-value products and fundamental studies. |
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| format | Article |
| id | doaj.art-bb62dd8afc8f42489347a5f0910d43eb |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T10:29:35Z |
| publishDate | 2021-06-01 |
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| spelling | doaj.art-bb62dd8afc8f42489347a5f0910d43eb2023-11-21T23:47:00ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-06-012212629910.3390/ijms22126299<i>Agrobacterium tumefaciens</i>-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—<i>Euglena gracilis</i>Ina Becker0Binod Prasad1Maria Ntefidou2Viktor Daiker3Peter Richter4Michael Lebert5Department of Cell Biology, University Erlangen-Nuremberg, 91058 Erlangen-Nuremberg, GermanyDepartment of Cell Biology, University Erlangen-Nuremberg, 91058 Erlangen-Nuremberg, GermanyDepartment of Cell Biology, University Erlangen-Nuremberg, 91058 Erlangen-Nuremberg, GermanyDepartment of Cell Biology, University Erlangen-Nuremberg, 91058 Erlangen-Nuremberg, GermanyDepartment of Cell Biology, University Erlangen-Nuremberg, 91058 Erlangen-Nuremberg, GermanyDepartment of Cell Biology, University Erlangen-Nuremberg, 91058 Erlangen-Nuremberg, Germany<i>Euglena gracilis</i> (<i>E. gracilis</i>) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of <i>E. gracilis</i> mediated by <i>Agrobacterium</i> (<i>A. tumefaciens</i>). <i>E. gracilis</i> was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. <i>A. tumefaciens</i>-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the <i>gus</i> gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the <i>gus</i> gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (<i>hptII</i>) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of <i>Euglena</i> with the <i>hptII-2A–gus</i> gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when <i>A. tumefaciens</i> LBA4404 (A<sub>600</sub> = 1.0) and <i>E. gracilis</i> (A<sub>750</sub> = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer <i>E. gracilis</i> for producing high-value products and fundamental studies.https://www.mdpi.com/1422-0067/22/12/6299<i>Euglena gracilis</i><i>Agrobacterium tumefaciens</i>transformationgenetic engineeringgene silencing |
| spellingShingle | Ina Becker Binod Prasad Maria Ntefidou Viktor Daiker Peter Richter Michael Lebert <i>Agrobacterium tumefaciens</i>-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—<i>Euglena gracilis</i> International Journal of Molecular Sciences <i>Euglena gracilis</i> <i>Agrobacterium tumefaciens</i> transformation genetic engineering gene silencing |
| title | <i>Agrobacterium tumefaciens</i>-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—<i>Euglena gracilis</i> |
| title_full | <i>Agrobacterium tumefaciens</i>-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—<i>Euglena gracilis</i> |
| title_fullStr | <i>Agrobacterium tumefaciens</i>-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—<i>Euglena gracilis</i> |
| title_full_unstemmed | <i>Agrobacterium tumefaciens</i>-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—<i>Euglena gracilis</i> |
| title_short | <i>Agrobacterium tumefaciens</i>-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—<i>Euglena gracilis</i> |
| title_sort | i agrobacterium tumefaciens i mediated nuclear transformation of a biotechnologically important microalga i euglena gracilis i |
| topic | <i>Euglena gracilis</i> <i>Agrobacterium tumefaciens</i> transformation genetic engineering gene silencing |
| url | https://www.mdpi.com/1422-0067/22/12/6299 |
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