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

Authors

Juan Ling, Huazhong Agricultural University
Renjie Li, Huazhong Agricultural University
Chinedu Charles Nwafor, Benson Idahosa University
Junluo Cheng, Huazhong Agricultural University
Maoteng Li, Huazhong University of Science and Technology
Qing Xi, Huazhong Agricultural University
Jian Wu, Yangzhou University
Lu Gan, Huazhong Agricultural University
Qingyong Yang, Huazhong Agricultural University
Chao Liu, Huazhong Agricultural University
Ming Chen, University of Nebraska - Lincoln
Yongming Zhou, Huazhong Agricultural University
Edgar B. Cahoon, University of Nebraska-LincolnFollow
Chunyu Zhang, Huazhong Agricultural UniversityFollow

Document Type

Article

Date of this Version

2018

Citation

Plant Biotechnology Journal (2018) 16, pp. 591–602, doi: 10.1111/pbi.12799.

Comments

Copyright 2017 The Authors. This is an open access article under the terms of the Creative Commons Attribution License.

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. 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.