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Title: Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana

Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs or multiple transformation events that is often restricted by a lack of available transformation markers. Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven non-family genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. In conclusion, we present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering.
Authors:
 [1] ;  [2] ;  [2] ;  [2] ;  [3] ;  [2] ;  [2]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; KWS SAAT SE, Einbeck (Germany)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
  3. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences
Publication Date:
Grant/Contract Number:
SC0008834; AC05-00OR22725
Type:
Accepted Manuscript
Journal Name:
Plant Methods
Additional Journal Information:
Journal Volume: 12; Journal Issue: 1; Journal ID: ISSN 1746-4811
Publisher:
BioMed Central
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 58 GEOSCIENCES
OSTI Identifier:
1294077
Alternate Identifier(s):
OSTI ID: 1311232

Czarnecki, Olaf, Bryan, Anthony C., Jawdy, Sara S., Yang, Xiaohan, Cheng, Zong-Ming, Chen, Jin-Gui, and Tuskan, Gerald A.. Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana. United States: N. p., Web. doi:10.1186/s13007-016-0116-8.
Czarnecki, Olaf, Bryan, Anthony C., Jawdy, Sara S., Yang, Xiaohan, Cheng, Zong-Ming, Chen, Jin-Gui, & Tuskan, Gerald A.. Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana. United States. doi:10.1186/s13007-016-0116-8.
Czarnecki, Olaf, Bryan, Anthony C., Jawdy, Sara S., Yang, Xiaohan, Cheng, Zong-Ming, Chen, Jin-Gui, and Tuskan, Gerald A.. 2016. "Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana". United States. doi:10.1186/s13007-016-0116-8. https://www.osti.gov/servlets/purl/1294077.
@article{osti_1294077,
title = {Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana},
author = {Czarnecki, Olaf and Bryan, Anthony C. and Jawdy, Sara S. and Yang, Xiaohan and Cheng, Zong-Ming and Chen, Jin-Gui and Tuskan, Gerald A.},
abstractNote = {Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs or multiple transformation events that is often restricted by a lack of available transformation markers. Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven non-family genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. In conclusion, we present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering.},
doi = {10.1186/s13007-016-0116-8},
journal = {Plant Methods},
number = 1,
volume = 12,
place = {United States},
year = {2016},
month = {2}
}

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