丝瓜多聚泛素基因(LcUBQ)的克隆及表达分析

Cloning and Expression Analysis of Polyubiquitin Gene (LcUBQ) in Luffa cylindrica

泛素是一种逆境响应蛋白,所介导的泛素/26S蛋白酶体途径在植物适应逆境过程中发挥重要作用。为探究丝瓜(Luffa cylindrica)泛素基因的功能,采用RT-PCR(reverse transcription-PCR)和RACE(rapid-amplification of cDNA ends)技术从丝瓜中克隆到多聚泛素基因(polyubiquitin gene),命名为LcUBQ(Gen Bank登录号:KR349345),该基因cDNA全长1 579 bp,包含一个1 371 bp的完整开放阅读框,编码457个氨基酸组成的蛋白质,预测其分子量和等电点分别为51.26 kDa和7.05,未发现信号肽和跨膜结构,WoLF PSORT预测其亚细胞定位于细胞质,二级结构中α-螺旋、延伸带和无规卷曲各占24.73%、22.54%和52.74%。进化树分析表明,丝瓜LcUBQ蛋白质与黄瓜、甜瓜等葫芦科植物亲缘关系较近。荧光定量PCR分析发现,LcUBQ基因在丝瓜的不同组织皆有表达,其中在根和叶中表达量最低,高温(40°C)、低温(4°C)以及弱光[20μmol/(m^2?s)]均能诱导叶片中LcUBQ基因的高表达,尤其是低温胁迫。低温胁迫下,LcUBQ基因的表达量呈现先升高再下降的趋势,与其对应的泛素水平的变化基本一致,并且无论是低温或弱光处理,LcUBQ基因的表达量及对应的泛素水平均在前2 h增加得最为明显,推测LcUBQ基因可能参与丝瓜的早期胁迫信号转导及低温、弱光胁迫应答过程。

Ubiquitin is a stress responses protein which mediates ubiquitin/26 S proteasome pathway and plays an important role in plant adaptation to various environmental stresses. To investigate the function of polyubiquitin gene in Luffa cylindrica, LcUBQ was isolated from luffa by using RACE（rapid-amplification of cDNA ends） and RT-PCR（reverse transcription-PCR） techniques（Gen Bank accession number was JN979372）. It was 1 579 bp, which contained a 1 371 bp open reading frame（ORF） that encoded 457 amino acids, with a predicted molecular weight of 51.26 kDa and a hypothetical isoelectric point of 7.05. This protein lacked the signal peptides and membrane-spanning domains and the WoLF PSORT protection indicated that it was located in cytoplasm. The secondary and tertiary protein structures were predicted, which consisted of 24.73% α-helices, 22.54% extended strands, and 52.74% random coils. Phylogenetic analysis illustrated that LcUBQ had high similarity to the UBQ of Cucurbitaceae plants such as Cucumis sativus and Cucumis melo. The results of fluorescence quantitative PCR analysis revealed that LcUBQ was expressed in roots, stems, leaves, flowers and fruits, and the lowest expression was found in roots and leaves. LcUBQ could be induced in leaves by high temperature（40 °C）, low temperature（4 °C） and low light（20 μmol/（m^2？s）） stresses, especially low temperature stress. Under low temperature stress, the expression of LcUBQ presented as an increased at first and then decreased trend, and the trend was consistent with the changes in the corresponding ubiquitin protein levels. Moreover, the levels of LcUBQ and ubiquitin protein were markedly elevated after stress for 2 h whether low temperature or low light. The results suggested that LcUBQ might be involved in early stress signal transduction and low temperature and low light stress response process in Luffa cylindrica.