Molecular and biochemical characterization of VR-EILs encoding Mung bean ETHYLENE INSENSITIVE3-LIKE proteins
Molecular and biochemical characterization of VR-EILs encoding Mung bean ETHYLENE INSENSITIVE3-LIKE proteins
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DOI：
10.1104/pp.103.022574
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发表时间：
2003-07-01
期刊：
影响因子：
7.4
通讯作者：
Kim, WT
中科院分区：
文献类型：
--
作者：
Lee, JH;Kim, WT
乙烯不敏感3 （EIN3）是拟南芥中参与乙烯信号转导途径的转录因子。通过逆转录聚合酶链反应和筛选绿豆下胚轴cDNA文库，分离到编码EIN3-LIKE蛋白的两个全长cDNA克隆pVR-EIL1和pVR-EIL2。VR-EIL1和VR-EIL2与先前从拟南芥、烟草（Nicotiana tabacum）和番茄（Lycopersicon esculentum）植物中分离到的EIN3同源物具有70%的同源性，并表现出不同程度的序列保守性（39%-65%）。凝胶阻滞实验显示，这两种VR-EIL蛋白都能与烟草TEIL蛋白的最佳结合序列-1特异性相互作用，且与VR-EIL蛋白2的结合效率高于与VR-EIL蛋白1的结合效率。利用VR-EIL::smGFP融合基因在洋葱表皮细胞中的瞬时表达分析表明，VR-EIL蛋白能有效靶向细胞核。VR-EIL2与GAL4 dna结合域的融合蛋白强烈激活酵母细胞中报告基因的转录，并且转录刺激活性的必要结构域位于由50个氨基酸残基组成的氨基末端酸性区域。与之前在过表达EIN3和teil的拟南芥植物中发现的情况相反，在花椰菜花叶病毒35S启动子的控制下，表达VR-EIL基因的转基因烟草幼苗没有表现出组成性的三重反应。相反，它们表现出根毛的增殖显著增强，这是典型的乙烯反应表型之一，并且对外源乙烯的敏感性增加。此外，编码β -1,3-葡聚糖酶、渗透素和PR1的致病相关（PR）基因在未添加乙烯的35S::VR-EIL中组成性表达，并在乙烯处理下被高诱导。这些结果表明，VR-EILs在烟草细胞中起作用，从而有效地反激活含有gc -box的PR基因并增强对乙烯的敏感性。本文讨论了VR-EILs可能的生理作用，认为它们是乙烯信号通路的活性成分，并且它们的异源表达构成了烟草植物乙烯反应的一个子集。
ETHYLENE INSENSITIVE3 (EIN3) is a transcription factor involved in the ethylene signal transduction pathway in Arabidopsis. Two full-length cDNA clones, pVR-EIL1 and pVR-EIL2, encoding EIN3-LIKE proteins were isolated by reverse transcriptase-polymerase chain reaction and by screening the cDNA library of mung bean (Vigna radiata) hypocotyls. VR-EIL1 and VR-EIL2 share 70% identity and display varying degrees of sequence conservation (39%-65%) with previously isolated EIN3 homologs from Arabidopsis, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) plants. Gel retardation assay revealed that both VR-EILs were able to interact specifically with optimal binding sequence-1, the recently identified optimal binding sequence for tobacco TEIL, with the binding of VR-EIL2 being more efficient than that of VR-EIL1 Transient expression analysis using a VR-EIL::smGFP fusion gene in onion (Allium cepa) epidermal cells indicated that the VR-EIL proteins were effectively targeted to the nucleus. The fusion protein of VR-EIL2 with GAL4 DNA-binding domain strongly activated transcription of a reporter gene in yeast cells, and an essential domain for transcription-stimulating activity was localized to the amino-terminal acidic region that consists of 50 amino acid residues. In contrast with what has been previously found in EIN3- and TEIL-overexpressing Arabidopsis plants, transgenic tobacco seedlings expressing the VR-EIL genes under the control of cauliflower mosaic virus 35S promoter did not exhibit a constitutive triple response. Instead, they displayed a markedly enhanced proliferation of root hairs, one of the typical ethylene response phenotypes, and increased sensitivity to exogenous ethylene. In addition, the pathogenesis-related (PR) genes encoding beta-1,3-glucanase, osmotin, and PR1 were constitutively expressed in 35S::VR-EIL lines without added ethylene, and were hyperinduced in response to ethylene treatment. These results indicate that VR-EILs are functional in tobacco cells, thereby effectively transactivating the GCC-box-containing PR genes and enhancing sensitivity to ethylene. The possible physiological role of VR-EILs is discussed in the light of the suggestion that they are active components of the ethylene-signa ling pathway and their heterologous expressions constitutively turn on a subset of ethylene responses in tobacco plants.