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Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters

In plant genetic engineering, the identification of gene promoters leading to particular expression patterns is crucial for the development of new genetically modified plant generations. This research was conducted in order to isolate and characterize several new promoters from cassava (Manihot escu...

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Autores principales: Suhandono, Sony, Apriyanto, Ardha, Ihsani, Nisa
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880305/
https://www.ncbi.nlm.nih.gov/pubmed/24404183
http://dx.doi.org/10.1371/journal.pone.0084692
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author Suhandono, Sony
Apriyanto, Ardha
Ihsani, Nisa
author_facet Suhandono, Sony
Apriyanto, Ardha
Ihsani, Nisa
author_sort Suhandono, Sony
collection PubMed
description In plant genetic engineering, the identification of gene promoters leading to particular expression patterns is crucial for the development of new genetically modified plant generations. This research was conducted in order to isolate and characterize several new promoters from cassava (Manihot esculenta Crantz) elongation factor 1 alpha (EF1A) gene family. Three promoters MeEF1A3, MeEF1A4 and MeEF1A5 were successfully isolated. Sequence analyses showed that all of the promoters contain three conserved putative cis-acting elements which are located upstream of the transcription start site. These elements are included a TEF1, a TELO and TATA boxes. In addition, all of the promoters also have the 5′UTR intron but with a different lengths. These promoters were constructed translationally with gusA reporter gene (promoter::gusA fusion) in pBI-121 binary vector to build a new binary vector using Overlap Extension PCR Cloning (OEPC) technique. Transient expression assay that was done by using agroinfiltration method was used to show functionality of these promoters. Qualitative and quantitative analysis from GUS assay showed that these promoters were functional and conferred a specific activity in tobacco seedlings (Nicotiana tabacum), tomato fruits (Solanum lycopersicum) and banana fruits (Musa acuminata). We hypothesized that MeEF1A6 could be categorized as a constitutive promoter because it was able to drive the gene expression in all transformed tissue described in here and also comparable to CaMV35S. On the other hand, MeEF1A3 drove specific expression in the aerial parts of seedlings such as hypocotyl and cotyledon thus MeEF1A5 drove specific expression in fruit tissue. The results obtained from transient analysis showed that these promoters had a distinct activity although they came from same gene family. The DNA sequences identified here are new promoters potentially use for genetic engineering in cassava or other plants.
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spelling pubmed-38803052014-01-08 Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters Suhandono, Sony Apriyanto, Ardha Ihsani, Nisa PLoS One Research Article In plant genetic engineering, the identification of gene promoters leading to particular expression patterns is crucial for the development of new genetically modified plant generations. This research was conducted in order to isolate and characterize several new promoters from cassava (Manihot esculenta Crantz) elongation factor 1 alpha (EF1A) gene family. Three promoters MeEF1A3, MeEF1A4 and MeEF1A5 were successfully isolated. Sequence analyses showed that all of the promoters contain three conserved putative cis-acting elements which are located upstream of the transcription start site. These elements are included a TEF1, a TELO and TATA boxes. In addition, all of the promoters also have the 5′UTR intron but with a different lengths. These promoters were constructed translationally with gusA reporter gene (promoter::gusA fusion) in pBI-121 binary vector to build a new binary vector using Overlap Extension PCR Cloning (OEPC) technique. Transient expression assay that was done by using agroinfiltration method was used to show functionality of these promoters. Qualitative and quantitative analysis from GUS assay showed that these promoters were functional and conferred a specific activity in tobacco seedlings (Nicotiana tabacum), tomato fruits (Solanum lycopersicum) and banana fruits (Musa acuminata). We hypothesized that MeEF1A6 could be categorized as a constitutive promoter because it was able to drive the gene expression in all transformed tissue described in here and also comparable to CaMV35S. On the other hand, MeEF1A3 drove specific expression in the aerial parts of seedlings such as hypocotyl and cotyledon thus MeEF1A5 drove specific expression in fruit tissue. The results obtained from transient analysis showed that these promoters had a distinct activity although they came from same gene family. The DNA sequences identified here are new promoters potentially use for genetic engineering in cassava or other plants. Public Library of Science 2014-01-03 /pmc/articles/PMC3880305/ /pubmed/24404183 http://dx.doi.org/10.1371/journal.pone.0084692 Text en © 2014 Suhandono et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Suhandono, Sony
Apriyanto, Ardha
Ihsani, Nisa
Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters
title Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters
title_full Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters
title_fullStr Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters
title_full_unstemmed Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters
title_short Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters
title_sort isolation and characterization of three cassava elongation factor 1 alpha (meef1a) promoters
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880305/
https://www.ncbi.nlm.nih.gov/pubmed/24404183
http://dx.doi.org/10.1371/journal.pone.0084692
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