A Synthetic Strong and Constitutive Promoter Derived from the <i>Stellaria media</i> pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants
Synthetic promoters are vital for genetic engineering-based strategies for crop improvement, but effective methodologies for their creation and systematic testing are lacking. We report here on the comparative analysis of the promoters pro-SmAMP1 and pro-SmAMP2 from <i>Stellaria media ANTIMICR...
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MDPI AG
2020-11-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/11/12/1407 |
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| author | Larisa N. Efremova Svetlana R. Strelnikova Guzel R. Gazizova Elena A. Minkina Roman A. Komakhin |
| author_facet | Larisa N. Efremova Svetlana R. Strelnikova Guzel R. Gazizova Elena A. Minkina Roman A. Komakhin |
| author_sort | Larisa N. Efremova |
| collection | DOAJ |
| description | Synthetic promoters are vital for genetic engineering-based strategies for crop improvement, but effective methodologies for their creation and systematic testing are lacking. We report here on the comparative analysis of the promoters pro-SmAMP1 and pro-SmAMP2 from <i>Stellaria media ANTIMICROBIAL PEPTIDE1</i> (<i>AMP1</i>) and <i>ANTIMICROBIAL PEPTIDE2</i> (<i>AMP2</i>). These promoters are more effective than the well-known <i>Cauliflower mosaic virus</i> 35S promoter. Although these promoters share about 94% identity, the pro-SmAMP1 promoter demonstrated stronger transient expression of a reporter gene in <i>Agrobacterium</i> infiltration of <i>Nicotiana benthamiana</i> leaves, while the pro-SmAMP2 promoter was more effective for the selection of transgenic tobacco (<i>Nicotiana tabacum</i>) cells when driving a selectable marker. Using the cap analysis of gene expression method, we detected no differences in the structure of the transcription start sites for either promoter in transgenic plants. For both promoters, we used fine-scale deletion analysis to identify 160 bp-long sequences that retain the unique properties of each promoter. With the use of chimeric promoters and directed mutagenesis, we demonstrated that the superiority of the pro-SmAMP1 promoter for <i>Agrobacterium</i>-mediated infiltration is caused by the proline-inducible ACTCAT <i>cis</i>-element strictly positioned relative to the TATA box in the core promoter. Surprisingly, the ACTCAT <i>cis</i>-element not only activated but also suppressed the efficiency of the pro-SmAMP1 promoter under proline stress. The absence of the ACTCAT <i>cis</i>-element and CAANNNNATC motif (negative regulator) in the pro-SmAMP2 promoter provided a more constitutive gene expression profile and better selection of transgenic cells on selective medium. We created a new synthetic promoter that enjoys high effectiveness both in transient expression and in selection of transgenic cells. Intact promoters with differing properties and high degrees of sequence identity may thus be used as a basis for the creation of new synthetic promoters for precise and coordinated gene expression. |
| first_indexed | 2024-03-10T14:33:28Z |
| format | Article |
| id | doaj.art-e0309d196a48461bbfc9cf919ca9d47d |
| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-10T14:33:28Z |
| publishDate | 2020-11-01 |
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| record_format | Article |
| series | Genes |
| spelling | doaj.art-e0309d196a48461bbfc9cf919ca9d47d2023-11-20T22:27:45ZengMDPI AGGenes2073-44252020-11-011112140710.3390/genes11121407A Synthetic Strong and Constitutive Promoter Derived from the <i>Stellaria media</i> pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in PlantsLarisa N. Efremova0Svetlana R. Strelnikova1Guzel R. Gazizova2Elena A. Minkina3Roman A. Komakhin4All-Russia Research Institute of Agricultural Biotechnology, Moscow 127550, RussiaAll-Russia Research Institute of Agricultural Biotechnology, Moscow 127550, RussiaInstitute of Fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, RussiaInstitute of Fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, RussiaAll-Russia Research Institute of Agricultural Biotechnology, Moscow 127550, RussiaSynthetic promoters are vital for genetic engineering-based strategies for crop improvement, but effective methodologies for their creation and systematic testing are lacking. We report here on the comparative analysis of the promoters pro-SmAMP1 and pro-SmAMP2 from <i>Stellaria media ANTIMICROBIAL PEPTIDE1</i> (<i>AMP1</i>) and <i>ANTIMICROBIAL PEPTIDE2</i> (<i>AMP2</i>). These promoters are more effective than the well-known <i>Cauliflower mosaic virus</i> 35S promoter. Although these promoters share about 94% identity, the pro-SmAMP1 promoter demonstrated stronger transient expression of a reporter gene in <i>Agrobacterium</i> infiltration of <i>Nicotiana benthamiana</i> leaves, while the pro-SmAMP2 promoter was more effective for the selection of transgenic tobacco (<i>Nicotiana tabacum</i>) cells when driving a selectable marker. Using the cap analysis of gene expression method, we detected no differences in the structure of the transcription start sites for either promoter in transgenic plants. For both promoters, we used fine-scale deletion analysis to identify 160 bp-long sequences that retain the unique properties of each promoter. With the use of chimeric promoters and directed mutagenesis, we demonstrated that the superiority of the pro-SmAMP1 promoter for <i>Agrobacterium</i>-mediated infiltration is caused by the proline-inducible ACTCAT <i>cis</i>-element strictly positioned relative to the TATA box in the core promoter. Surprisingly, the ACTCAT <i>cis</i>-element not only activated but also suppressed the efficiency of the pro-SmAMP1 promoter under proline stress. The absence of the ACTCAT <i>cis</i>-element and CAANNNNATC motif (negative regulator) in the pro-SmAMP2 promoter provided a more constitutive gene expression profile and better selection of transgenic cells on selective medium. We created a new synthetic promoter that enjoys high effectiveness both in transient expression and in selection of transgenic cells. Intact promoters with differing properties and high degrees of sequence identity may thus be used as a basis for the creation of new synthetic promoters for precise and coordinated gene expression.https://www.mdpi.com/2073-4425/11/12/1407synthetic promoterGUSACTCAT cis-element<i>nptII</i>genetic transformationgene expression |
| spellingShingle | Larisa N. Efremova Svetlana R. Strelnikova Guzel R. Gazizova Elena A. Minkina Roman A. Komakhin A Synthetic Strong and Constitutive Promoter Derived from the <i>Stellaria media</i> pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants Genes synthetic promoter GUS ACTCAT cis-element <i>nptII</i> genetic transformation gene expression |
| title | A Synthetic Strong and Constitutive Promoter Derived from the <i>Stellaria media</i> pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants |
| title_full | A Synthetic Strong and Constitutive Promoter Derived from the <i>Stellaria media</i> pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants |
| title_fullStr | A Synthetic Strong and Constitutive Promoter Derived from the <i>Stellaria media</i> pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants |
| title_full_unstemmed | A Synthetic Strong and Constitutive Promoter Derived from the <i>Stellaria media</i> pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants |
| title_short | A Synthetic Strong and Constitutive Promoter Derived from the <i>Stellaria media</i> pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants |
| title_sort | synthetic strong and constitutive promoter derived from the i stellaria media i pro smamp1 and pro smamp2 promoters for effective transgene expression in plants |
| topic | synthetic promoter GUS ACTCAT cis-element <i>nptII</i> genetic transformation gene expression |
| url | https://www.mdpi.com/2073-4425/11/12/1407 |
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