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Development of iFOX‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid Brassica napus

Functional genomic studies of many polyploid crops, including rapeseed (Brassica napus), are constrained by limited tool sets. Here we report development of a gain‐of‐function platform, termed ‘iFOX (inducible Full‐length cDNA OvereXpressor gene)‐Hunting’, for inducible expression of B. napus seed c...

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Autores principales: Ling, Juan, Li, Renjie, Nwafor, Chinedu Charles, Cheng, Junluo, Li, Maoteng, Xu, Qing, Wu, Jian, Gan, Lu, Yang, Qingyong, Liu, Chao, Chen, Ming, Zhou, Yongming, Cahoon, Edgar B., Zhang, Chunyu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787830/
https://www.ncbi.nlm.nih.gov/pubmed/28718508
http://dx.doi.org/10.1111/pbi.12799
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author Ling, Juan
Li, Renjie
Nwafor, Chinedu Charles
Cheng, Junluo
Li, Maoteng
Xu, Qing
Wu, Jian
Gan, Lu
Yang, Qingyong
Liu, Chao
Chen, Ming
Zhou, Yongming
Cahoon, Edgar B.
Zhang, Chunyu
author_facet Ling, Juan
Li, Renjie
Nwafor, Chinedu Charles
Cheng, Junluo
Li, Maoteng
Xu, Qing
Wu, Jian
Gan, Lu
Yang, Qingyong
Liu, Chao
Chen, Ming
Zhou, Yongming
Cahoon, Edgar B.
Zhang, Chunyu
author_sort Ling, Juan
collection PubMed
description Functional genomic studies of many polyploid crops, including rapeseed (Brassica napus), are constrained by limited tool sets. Here we report development of a gain‐of‐function platform, termed ‘iFOX (inducible Full‐length cDNA OvereXpressor gene)‐Hunting’, for inducible expression of B. napus seed cDNAs in Arabidopsis. A Gateway‐compatible plant gene expression vector containing a methoxyfenozide‐inducible constitutive promoter for transgene expression was developed. This vector was used for cloning of random cDNAs from developing B. napus seeds and subsequent Agrobacterium‐mediated transformation of Arabidopsis. The inducible promoter of this vector enabled identification of genes upon induction that are otherwise lethal when constitutively overexpressed and to control developmental timing of transgene expression. Evaluation of a subset of the resulting ~6000 Arabidopsis transformants revealed a high percentage of lines with full‐length B. napus transgene insertions. Upon induction, numerous iFOX lines with visible phenotypes were identified, including one that displayed early leaf senescence. Phenotypic analysis of this line (rsl‐1327) after methoxyfenozide induction indicated high degree of leaf chlorosis. The integrated B. napus cDNA was identified as a homolog of an Arabidopsis acyl‐CoA binding protein (ACBP) gene designated BnACBP1‐like. The early senescence phenotype conferred by BnACBP1‐like was confirmed by constitutive expression of this gene in Arabidopsis and B. napus. Use of the inducible promoter in the iFOX line coupled with RNA‐Seq analyses allowed mechanistic clues and a working model for the phenotype associated with BnACBP1‐like expression. Our results demonstrate the utility of iFOX‐Hunting as a tool for gene discovery and functional characterization of Brassica napus genome.
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spelling pubmed-57878302018-02-05 Development of iFOX‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid Brassica napus Ling, Juan Li, Renjie Nwafor, Chinedu Charles Cheng, Junluo Li, Maoteng Xu, Qing Wu, Jian Gan, Lu Yang, Qingyong Liu, Chao Chen, Ming Zhou, Yongming Cahoon, Edgar B. Zhang, Chunyu Plant Biotechnol J Research Articles Functional genomic studies of many polyploid crops, including rapeseed (Brassica napus), are constrained by limited tool sets. Here we report development of a gain‐of‐function platform, termed ‘iFOX (inducible Full‐length cDNA OvereXpressor gene)‐Hunting’, for inducible expression of B. napus seed cDNAs in Arabidopsis. A Gateway‐compatible plant gene expression vector containing a methoxyfenozide‐inducible constitutive promoter for transgene expression was developed. This vector was used for cloning of random cDNAs from developing B. napus seeds and subsequent Agrobacterium‐mediated transformation of Arabidopsis. The inducible promoter of this vector enabled identification of genes upon induction that are otherwise lethal when constitutively overexpressed and to control developmental timing of transgene expression. Evaluation of a subset of the resulting ~6000 Arabidopsis transformants revealed a high percentage of lines with full‐length B. napus transgene insertions. Upon induction, numerous iFOX lines with visible phenotypes were identified, including one that displayed early leaf senescence. Phenotypic analysis of this line (rsl‐1327) after methoxyfenozide induction indicated high degree of leaf chlorosis. The integrated B. napus cDNA was identified as a homolog of an Arabidopsis acyl‐CoA binding protein (ACBP) gene designated BnACBP1‐like. The early senescence phenotype conferred by BnACBP1‐like was confirmed by constitutive expression of this gene in Arabidopsis and B. napus. Use of the inducible promoter in the iFOX line coupled with RNA‐Seq analyses allowed mechanistic clues and a working model for the phenotype associated with BnACBP1‐like expression. Our results demonstrate the utility of iFOX‐Hunting as a tool for gene discovery and functional characterization of Brassica napus genome. John Wiley and Sons Inc. 2017-08-22 2018-02 /pmc/articles/PMC5787830/ /pubmed/28718508 http://dx.doi.org/10.1111/pbi.12799 Text en © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
Ling, Juan
Li, Renjie
Nwafor, Chinedu Charles
Cheng, Junluo
Li, Maoteng
Xu, Qing
Wu, Jian
Gan, Lu
Yang, Qingyong
Liu, Chao
Chen, Ming
Zhou, Yongming
Cahoon, Edgar B.
Zhang, Chunyu
Development of iFOX‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid Brassica napus
title Development of iFOX‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid Brassica napus
title_full Development of iFOX‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid Brassica napus
title_fullStr Development of iFOX‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid Brassica napus
title_full_unstemmed Development of iFOX‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid Brassica napus
title_short Development of iFOX‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid Brassica napus
title_sort development of ifox‐hunting as a functional genomic tool and demonstration of its use to identify early senescence‐related genes in the polyploid brassica napus
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787830/
https://www.ncbi.nlm.nih.gov/pubmed/28718508
http://dx.doi.org/10.1111/pbi.12799
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