Cloning and expression analysis of a U-box gene of E3 ubiquitin ligase from Medicago truncatula

doi:10.11686/cyxb2016073

Abstract

Abstract: The E3 ubiquitin ligase is essential for the specific recognition of target proteins of ubiquitin, and therefore, it plays an important role in plant growth and development. The U-box family of E3 ubiquitin ligases function in regulating plant immune responses and stress resistance. To the best of our knowledge, there have been no reports on U-box family genes in Medicago truncatula. In this study, a U-box gene (MtPUB4), consisting of a 2448-bp cDNA encoding a putative protein of 815 amino acids, was cloned from M. truncatula. A sequence alignment analysis revealed that the MtPUB4 protein contains a U-box domain (C-X2-H-X7-C-X7-C-X2-C-H-X2-H) and two ARM (Armadillo) regions. Therefore, it was classified as a U-box/ARM E3 ubiquitin-ligase. We constructed a prokaryotic expression vector (pET-MtPUB4), and an SDS-PAGE analysis confirmed that the MtPUB4 protein was successfully expressed in prokaryotic cells. Real-time polymerase chain reaction analyses showed that the transcript levels of MtPUB4 were higher in flowers and lower in roots. Abiotic stresses such as NaCl, polyethylene glycol, and abscisic acid resulted in increased transcript levels of MtPUB4, with higher transcript levels after longer induction times. The transcript levels of MtPUB4 decreased slightly under low-temperature stress. These results indicated that MtPUB4 may be involved in regulating growth, development, and stress resistance in M. truncatula. The construction of the pBI121-MtPUB4 vector has laid the foundation for transformation of Arabidopsis in future studies.

SHAO Lin-Hui, ZHENG Xing-Wei, LI Cong. Cloning and expression analysis of a U-box gene of E3 ubiquitin ligase from Medicago truncatula[J]. Acta Prataculturae Sinica, 2016, 25(7): 62-72.

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