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 |
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| Authors: | Chinedu Charles Nwafor Junluo Cheng Maoteng Li Qing Xu Jian Wu Lu Gan Qingyong Yang Chao Liu Ming Chen Yongming Zhou Edgar B Cahoon Chunyu Zhang |
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| Affiliation: | 1. National Research Centre of Rapeseed Engineering and Technology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China;2. Department of Crop Science, Benson Idahosa University, Benin City, Nigeria;3. Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China;4. Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou, China;5. Center for Plant Science Innovation and Department of Biochemistry, University of Nebraska‐Lincoln, Lincoln, NE, USA |
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| Abstract: | 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. napuscDNA 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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| Keywords: | iFOX‐Hunting system functional genomic platform
Brassica napus
Arabidopsis thaliana
early leaf senescence
BnACBP1‐like
acyl‐CoA binding protein |
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