Abstract
The EARLY FLOWERING 4 (ELF4) gene has been reported as a requirement for photoperiod perception and circadian function. In this study, an ELF4 homolog gene EARLY FLOWERING 4-like4 (EFL4) was isolated from Doritaenopsis “Tinny Tender” (Doritaenopsis Happy Smile × Happy Valentine) and designated as DhEFL4. The acquired DhEFL4 cDNA was 593 bp with a 345 bp open reading frame encoding 114 amino acids. The putative DhEFL4 protein contained a conserved region, DUF1313, which is shared by the ELF4 family. Amino acid sequence alignment showed that DhEFL4 shared high similarity to ELF4 and ELF4-like homologs from other species. The DhEFL4 promoter was also obtained, and 241 cis-elements were found. The diurnal expression of DhEFL4, which may be affected by the EVENINGAT (EE) and circadian motif was analyzed by quantitative PCR under short- and long-day conditions. Reverse-transcription PCR analysis results showed that DhEFL4 is an evening-expressed circadian gene. DhEFL4 was expressed in both vegetative and reproductive organs, and its transcripts reached higher levels in the buds. To further study the function of the DhEFL4 gene, DhEFL4 was overexpressed in Arabidopsis thaliana by Agrobacterium-mediated transformation. The flowering of these transgenic plants was more delayed than that of wild-type Arabidopsis by 12−14 days, and flower stems increased. Results indicated that DhEFL4 may perform an important function in the regulation of flowering.
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Abbreviations
- ELF4:
-
EARLY FLOWERING 4
- EFL4:
-
EARLY FLOWERING 4-LIKE4
- EE:
-
EVENINGAT
- SD:
-
Short day
- LD:
-
Long day
- CCA1:
-
CIRCADIAN CLOCK ASSOCIATED 1
- LHY:
-
LATE ELONGATED HYPOCOTYL
- TOC1:
-
TIMING OF CAB EXPRESSION 1
- ELF3:
-
EARLY FLOWERING 3
- LUX:
-
LUX ARRHYTHMO
- CaMV:
-
Cauliflower mosaic virus
- EST:
-
Expressed sequence tag
- Lhc:
-
Light-harvesting complex protein genes
- Lhc b:
-
Light-harvesting chlorophyll a/b binding protein genes of PSII
- CO:
-
CONSTANS
- RACE:
-
Rapid amplification of cDNA ends
- ZT:
-
Zeitgeber time
- qRT-PCR:
-
Quantitative real-time PCR
- PCR:
-
Polymerase chain reaction
- MeJA:
-
Methyl jasmonate
- MS:
-
Murashige and Skoog
- ORF:
-
Open reading frame
- RT-PCR:
-
Reverse transcription-polymerase chain reaction
- WT:
-
Wild-type
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Acknowledgments
We thank Dr. Zhi John Lu (MOE Key of Bioinformatics, School of Life Science, Tsinghua University) for the useful suggestions on improving the manuscript. This study was supported by the National Natural Science Foundation of China (Grant Nos. 30771762, 31170658, and 31301811) and the Science & Technology Key Project of Zhejiang Province (Grant No. 2012C12909-11).
Authors’ Contribution
W. Chen designed, performed, and managed the research work and wrote the manuscript. Q. Qin improved the manuscript. Y. Zheng and C. Wang supplied the materials. S. Wang analyzed some data. M. Zhou, C. Zhang, and Y. Cui managed the experiment partly.
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The authors declare that they have no conflict of interest.
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Electronic Supplementary Material
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Online Resource 1
Sequence information on alignment of the DhEFL4 amino acid sequences with ELF4 and ELF4-like from different plant species. (DOCX 16 kb)
Online Resource 2
Sequence information on phylogenetic analysis of the DhEFL4 encoded amino acid sequences from different plant species. (DOCX 83 kb)
Online Resource 3
Nucleotide and deduced amino acid sequences of DhEFL4 cDNA. The DUF1313 superfamily region is indicated with a parenthesis in bold. (GIF 21 kb)
Online Resource 4
Putative promoter region sequence and its transcription factor binding motifs, as predicted from PLACE and PlantCARE. (GIF 138 kb)
Online Resource 5
Cis-acting regulatory elements analysis of promoter sequences by PLACE and PlantCARE. (DOCX 24 kb)
Table S1
Sequences of the primers used in this study. (DOCX 132 kb)
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Chen, W., Qin, Q., Zheng, Y. et al. Overexpression of Doritaenopsis Hybrid EARLY FLOWERING 4-like4 Gene, DhEFL4, Postpones Flowering in Transgenic Arabidopsis . Plant Mol Biol Rep 34, 103–117 (2016). https://doi.org/10.1007/s11105-015-0899-1
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DOI: https://doi.org/10.1007/s11105-015-0899-1