李铁铮, 郑一哲, 戎玉清, 魏胜利, 王晓晖, 屠鹏飞. 白木香AsERF1基因的克隆、亚细胞定位与表达分析J. 药学学报, 2020,55(8): 1957-1964. doi: 10.16438/j.0513-4870.2020-0176
引用本文: 李铁铮, 郑一哲, 戎玉清, 魏胜利, 王晓晖, 屠鹏飞. 白木香AsERF1基因的克隆、亚细胞定位与表达分析J. 药学学报, 2020,55(8): 1957-1964. doi: 10.16438/j.0513-4870.2020-0176
LI Tie-zheng, ZHENG Yi-zhe, RONG Yu-qing, WEI Sheng-li, WANG Xiao-hui, TU Peng-fei. Gene cloning, subcellular localization and expression analysis of the AsERF1 gene from Aquilaria sinensisJ. Acta Pharmaceutica Sinica, 2020,55(8): 1957-1964. doi: 10.16438/j.0513-4870.2020-0176
Citation: LI Tie-zheng, ZHENG Yi-zhe, RONG Yu-qing, WEI Sheng-li, WANG Xiao-hui, TU Peng-fei. Gene cloning, subcellular localization and expression analysis of the AsERF1 gene from Aquilaria sinensisJ. Acta Pharmaceutica Sinica, 2020,55(8): 1957-1964. doi: 10.16438/j.0513-4870.2020-0176

白木香AsERF1基因的克隆、亚细胞定位与表达分析

Gene cloning, subcellular localization and expression analysis of the AsERF1 gene from Aquilaria sinensis

  • 摘要: 乙烯响应因子(ethylene-response factor,ERF)是AP2/ERF家族中的亚家族,在植物信号转导、生长发育和植物防御反应中起到重要作用。本研究以白木香(Aquilaria sinensis)为材料,设计特异性引物,克隆了白木香的AsERF1基因的cDNA序列,并进行氨基酸序列分析、原核表达和纯化、亚细胞定位、组织特异性分析、非生物胁迫诱导表达分析研究。白木香AsERF1基因的开放阅读框(ORF)长691 bp,编码229个氨基酸,其蛋白分子质量为25.36 kD。AsERF1蛋白含有1个保守的AP2区域,系统进化树分析表明AsERF1与毛果杨的ERF2蛋白亲缘性最高。利用本生烟瞬时表达系统研究表明AsERF1主要定位于植物细胞核中。构建原核表达载体pET28a-AsERF1,并在大肠杆菌BL21(DE3)菌株中成功表达AsERF1重组蛋白,纯化得到可溶性重组蛋白。实时荧光定量PCR结果显示AsERF1在叶中的表达量最高,茎次之,根和茎尖最低。盐、干旱、低温和重金属胁迫能够诱导AsERF1的表达,其中干旱胁迫对AsERF1的表达水平影响最显著。本研究为揭示ERF基因在白木香防御反应和沉香形成过程中的作用机制奠定基础。

     

    Abstract: Ethylene-response factors, which are a subfamily of the AP2/ERF family, play an important role in ethylene signal transduction, plant growth and plant resistant. In this study, a full-length cDNA of the AsERF1 gene was cloned from Aquilaria sinensis. Sequence analysis, prokaryotic expression and purification, subcellular localization, tissue-specific analysis and expression analysis under different abiotic stresses was performed. The open reading frame (ORF) of the AsERF1 gene was 691 bp, encoding a protein of 229 amino acids with a predicted molecular mass of 25.36 kD. The AsERF1 protein contained the conserved AP2 sequence of ERF protein. A phylogenetic analysis indicated that the AsERF1 protein showed greatest sequence similarity with ERF2 from Populus trichocarpa. The recombinant AsERF1 protein was expressed in Escherichia coli BL21(DE3) cells using the prokaryotic expression vector pET28a-AsERF1 and the recombinant AsERF1 protein was purified. Agrobacterium-mediated protein expression experiments demonstrated that AsERF1 mainly localized to the nucleus. Expression analysis indicated that AsERF1 was primarily observed in leaves. The AsERF1 expression level was induced by salt, drought, low temperature and CdCl2 treatment, while the abundance of AsERF1 was most significantly induced by drought stress. These results provide valuable insights into the role of AsERF1 in plant defense and the mechanism of agarwood formation.

     

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