蒺藜苜蓿E3泛素连接酶U-box基因克隆及表达分

doi:10.11686/cyxb2016073

摘要/Abstract

摘要： 泛素化在植物生长和发育过程中起重要作用,E3泛素连接酶负责对靶蛋白特异性识别,其中U-box结构的E3泛素连接酶具有调控植物生长发育和免疫反应等功能,并在抗逆性方面发挥作用。目前,关于蒺藜苜蓿U-box家族基因的研究尚未见报道。本研究从蒺藜苜蓿中克隆得到U-box (MtPUB4)基因,该基因cDNA全长2448 bp,编码815个氨基酸,包括1个U-box结构(C-X2-H-X7-C-X7-C-X2-C-H-X2-H)和5个ARM结构域,属于U-box/ARM类型E3泛素连接酶。构建原核表达载体pET-MtPUB4,通过聚丙烯酰胺凝胶电泳(SDS-PAGE)表明在大肠杆菌中表达了MtPUB4蛋白。荧光定量PCR分析表明MtPUB4基因在蒺藜苜蓿花中的表达量最高,在根中表达量最低。MtPUB4基因受到NaCl、聚乙二醇、脱落酸诱导,随着诱导时间增加,表达量呈现出增长趋势。这些结果表明,MtPUB4基因在蒺藜苜蓿生长发育和抗逆性中可能起到重要调节作用,但在低温胁迫中表达量稍微下降,变化不明显。本研究成功构建了pBI121-MtPUB4植物表达载体,为进一步转化拟南芥突变体Atpub4奠定了基础。

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.

邵麟惠, 郑兴卫, 李聪. 蒺藜苜蓿E3泛素连接酶U-box基因克隆及表达分[J]. 草业学报, 2016, 25(7): 62-72.

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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