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Characteristics and Function of a GmDREB5-Interacting Protein GmUBC13 in Soybean

XU Dong-bei1,2, YU Yue-hua3, HAN Qiao-ling2, MA Ya-nan1,2, GAO Shi-qing4, TIAN Ye2, XU Zhao-shi2, LI Lian-cheng2, QU Yan-ying3, MA You-zhi2, CHEN Ming2, CHEN Yao-feng1   

  1. 1College of Agronomy, Northwest A &F University, Yangling 712100, Shaanxi; 
    2    Institute of Crop Sciences, Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement/Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Beijing 100081; 
    3    College of Agronomy, Xinjiang Agricultural University, Urumqi 830052; 
    4    Beijing Hybrid Wheat Engineering and Technology Research Center, Beijing 100097
  • Received:2014-03-12 Revised:2014-05-14 Online:2014-09-16 Published:2014-09-16

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Abstract: 【Objective】The objectives of this research are to identify the interacting proteins of stress related DREB like transcription factor, GmDREB5 in soybean (Glycine max), analyze the characteristics and biological function of its interaction protein, GmUBC13, and study the regulation mechanism that GmDREB5 enhanced the stress tolerance in plants. 【Method】 By yeast two hybrid system, AP2 domain of GmDREB5 was used as a bait to screen the interacting proteins of GmDREB5 from the drought-treated soybean cDNA library, and further interaction between GmDREB5 and its candidate interacting protein was confirmed through interacting testing in yeast and pull-down assay in vitro, then, the properties of interacting protein, GmUBC13, such as phylogenetic tree, protein structure, subcellular localization were analyzed; expression profiles of GmUBC13 were tested under drought, salt, cold and ABA treatment by RT-PCR, and biological functions of GmUBC13 were also identified through transformation of tobacco in this research. 【Result】A GmDREB5-interacting protein, GmUBC13, belonging to the ubiquitin conjugating enzyme protein family, was obtained via screening drought-treated soybean cDNA library, and GmUBC13 contained UBCc (Ubiquitin-conjugating enzyme catalytic domain) domain, highly conserved E3 interaction residues and cysteine catalytic sites. Phylogenetic analyses showed that GmUBC13 shared the high homology with two members of the XV subgroup (containing total sixteen members) of ubiquitin conjugating enzyme like protein family, AtUBC13A (99%) and AtUBC13B (97%) in Arabidopsis and a ubiquitin conjugating enzyme like protein, Os01g0673600 (97%) in rice (Oryza sativa). Interaction between GmDREB5 and GmUBC13 was confirmed by interaction test in yeast and pull-down assay in vitro. The expression of GmUBC13 gene in soybean was analyzed by drought, high salt, low temperature and ABA treatment. After ABA application, the transcript of GmUBC13 was induced after 1 h and reached the peak at 10 h, and slight down-regulated at 24 h. Under drought- and salt-treatment, the transcript of GmUBC13 was induced after 1 h and gradually increased along with the treatment time, and reached the peak at 24 h; under the low temperature, the expression of GmUBC13 induced rapidly and reached the peak at 5 h, and there was no expression at 10 h and 24 h. Further subcellular localization analysis showed that GmDREB5 was located on the nuclear and cytoplasm, and GmUBC13 was located on the nuclear. The analysis result of gene function showed that there was no difference among transgenic tobacco lines GmUBC13-1, GmUBC13-2 and wild type (W38) under normal MS medium, whereas exposed to different concentrations of PEG, the chlorophyll contents decreased, and the length of root and segment above ground were inhibited in all plants. However, the chlorophyll contents were higher, and the length of root and segment above ground was longer in GmDREB13 transgenic plants compared with W38, and there was a significant difference between transgenic lines GmUBC13-2 and W38 under treatment of 8% PEG. For the drought tolerance assay, two-week old transgenic plants and W38 were transferred into soil and withheld water for 21 days, and then re-watered for 6 days. Final results showed that transgenic lines grew healthier than W38, and the survival rates of transgenic lines were significantly higher than W38, suggesting that overexpression of GmUBC13 enhanced drought tolerance in transgenic plant.【Conclusion】 Soybean ubiquitin conjugating enzyme, GmUBC13 interacted with stress related transcription factor GmDREB5, and GmUBC13 was induced by various stress treatments, and overexpression of GmUBC13 enhanced drought tolerance in transgenic tobacco plants.

Key words: soybean, DREB like transcription factor gene, ubiquitin-conjugating enzyme, interacting protein, gene function

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