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GmRFP1 encodes a previously unknown RING-type E3 ubiquitin ligase in Soybean (Glycine max)

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Abstract

RING-finger proteins with E3 ubiquitin ligase activity play important roles in the regulation of plant growth and development. In this study, a cDNA clone encoding a novel RING-finger protein, designated as GmRFP1, was isolated and characterized from soybean. GmRFP1 was an intronless gene encoding a predicted protein product of 392 amino acid residues with a molecular mass of ~43 kDa. The protein contained a RING-H2 motif and an N-terminal transmembrane domain. The transcript was observed in all detected organs and was up-regulated by abscisic acid (ABA) and salt stress, but down-regulated by cold and drought treatments. We further expressed and purified both wild type and mutant version of GmRFP1 in E. coli. In vitro assays showed that the purified GmRFP1 induced the formation of polyubiquitin chains while mutation within the RING-finger region abolished the ubiquitination activity. These findings suggest that GmRFP1 is a previously unknown E3 ubiquitin ligase in soybean and that the RING domain is required for its activity. It may play unappreciated roles in ABA signaling and stress responses via mediating the ubiquitination and degradation of target proteins through the ubiquitin-proteasome pathway.

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Abbreviations

RING:

Really interesting new gene

GmRFP1:

Glycine max RING-finger protein 1

Ub:

Ubiquitin

E1:

Ubiquitin-activating enzyme

E2:

Ubiquitin-conjugating enzyme

E3:

Ubiquitin-protein ligase

HECT:

Homology to E6-AP carboxy terminus

SCF:

Skp1-Cullin-F-box

APC:

Anaphase-promoting complex

ABA:

Abscisic acid

SAM:

Shoot apical meristem

DAF:

Days after flowering

RT:

Reverse transcription

qRT-PCR:

Quantitative real-time polymerase chain reaction

RACE:

Rapid amplification of cDNA ends

PEG:

Polyethylene glycol

GST:

Glutathione S-transferase

IPTG:

Isopropyl-1-thio-β-D-galactopyranoside

PBS:

Phosphate buffered saline

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Acknowledgments

We thank Dr. Fei Li (University of California, Berkeley, CA) for revising the manuscript, Dr. Aifen Zhou (University of Oklahoma, Norman, OK) for helpful suggestions and valuable discussions. We also thank Wenbiao Shen (Nanjing Agriculture University, Nanjing, China) for technical advice. This work was supported in part by National 973 Projects (no. 2004CB117206, no. 2002CB111304), National 863 Projects (no. 2006AA10Z1C1, 2007AA10Z193, no. 2006AA10A111), National Natural Science Foundation of China (no. 30490250, no. 30771362), the Specific Grant from the Ministry of Agriculture (no. 200803060), and the 111 Project from the Ministry of Education (B08025).

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  1. National Center for Soybean Improvement, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, 210095, Nanjing, People’s Republic of China

    Qiu-Li Du, Wen-Zhuo Cui, Chun-Hong Zhang & De-Yue Yu

  2. Department of Biology, Jining University, 273155, Jining, People’s Republic of China

    Qiu-Li Du

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  1. Qiu-Li Du
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Du, QL., Cui, WZ., Zhang, CH. et al. GmRFP1 encodes a previously unknown RING-type E3 ubiquitin ligase in Soybean (Glycine max). Mol Biol Rep 37, 685 (2010). https://doi.org/10.1007/s11033-009-9535-1

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