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Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance

Plant viruses impose serious threats on crop production. Artificial miRNAs can mediate specific and effective gene silencing in plants and are widely used in plant gene function studies and to engineer plant viral resistance. To facilitate the design of artificial miRNA genes, we developed a webserv...

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Autores principales: Yasir, Muhammad, Motawaa, Mohamed, Wang, Qingwei, Zhang, Xi, Khalid, Annum, Cai, Xingkui, Li, Feng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9412884/
https://www.ncbi.nlm.nih.gov/pubmed/36015429
http://dx.doi.org/10.3390/plants11162125
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author Yasir, Muhammad
Motawaa, Mohamed
Wang, Qingwei
Zhang, Xi
Khalid, Annum
Cai, Xingkui
Li, Feng
author_facet Yasir, Muhammad
Motawaa, Mohamed
Wang, Qingwei
Zhang, Xi
Khalid, Annum
Cai, Xingkui
Li, Feng
author_sort Yasir, Muhammad
collection PubMed
description Plant viruses impose serious threats on crop production. Artificial miRNAs can mediate specific and effective gene silencing in plants and are widely used in plant gene function studies and to engineer plant viral resistance. To facilitate the design of artificial miRNA genes, we developed a webserver, AMIRdesigner, which can be used to design oligos for artificial miRNA synthesis using wild-type and permutated MIR171 and MIR164 backbones. The artificial miRNA genes designed by AMIRdesigner can be easily assembled into miRNA clusters for multiple target sites. To validate the server functionality, we designed four artificial miRNA genes targeting four conserved regions in the potato leafroll virus genome using AMIRdesigner. These genes were synthesized with the server-designed oligos and further assembled into a quadruple miRNA cluster, which was cloned into an overexpression vector and transformed into potato plants. Small RNA Northern blot and virus inoculation analyses showed that a high level of artificial miRNA expression and good viral resistance were achieved in some of the transgenic lines. These results demonstrate the utility of our webserver AMIRdesigner for engineering crop viral resistance.
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spelling pubmed-94128842022-08-27 Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance Yasir, Muhammad Motawaa, Mohamed Wang, Qingwei Zhang, Xi Khalid, Annum Cai, Xingkui Li, Feng Plants (Basel) Article Plant viruses impose serious threats on crop production. Artificial miRNAs can mediate specific and effective gene silencing in plants and are widely used in plant gene function studies and to engineer plant viral resistance. To facilitate the design of artificial miRNA genes, we developed a webserver, AMIRdesigner, which can be used to design oligos for artificial miRNA synthesis using wild-type and permutated MIR171 and MIR164 backbones. The artificial miRNA genes designed by AMIRdesigner can be easily assembled into miRNA clusters for multiple target sites. To validate the server functionality, we designed four artificial miRNA genes targeting four conserved regions in the potato leafroll virus genome using AMIRdesigner. These genes were synthesized with the server-designed oligos and further assembled into a quadruple miRNA cluster, which was cloned into an overexpression vector and transformed into potato plants. Small RNA Northern blot and virus inoculation analyses showed that a high level of artificial miRNA expression and good viral resistance were achieved in some of the transgenic lines. These results demonstrate the utility of our webserver AMIRdesigner for engineering crop viral resistance. MDPI 2022-08-15 /pmc/articles/PMC9412884/ /pubmed/36015429 http://dx.doi.org/10.3390/plants11162125 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yasir, Muhammad
Motawaa, Mohamed
Wang, Qingwei
Zhang, Xi
Khalid, Annum
Cai, Xingkui
Li, Feng
Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance
title Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance
title_full Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance
title_fullStr Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance
title_full_unstemmed Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance
title_short Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance
title_sort simple webserver-facilitated method to design and synthesize artificial mirna gene and its application in engineering viral resistance
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9412884/
https://www.ncbi.nlm.nih.gov/pubmed/36015429
http://dx.doi.org/10.3390/plants11162125
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