Identification and analysis of the mechanism underlying heat-inducible expression of rice aconitase 1
Identification and analysis of the mechanism underlying heat-inducible expression of rice aconitase 1
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水稻乌头酸酶1热诱导表达机制鉴定及分析
DOI：
10.1016/j.plantsci.2015.01.003
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发表时间：
2015-04-01
期刊：
影响因子：
5.2
通讯作者：
Yang, Jian-Bo
中科院分区：
文献类型：
--
作者：
Li, Juan;Qin, Rui-Ying;Yang, Jian-Bo
呼吸代谢是植物适应逆境的一个重要方面，但人们对其了解甚少。顺乌头酸酶是三羧酸循环（TCA）的一个组成部分，其翻译后修饰可能参与胁迫耐受。然而，这种与胁迫相关的转录调控及其机制仍不清楚。在本研究中，我们发现，水稻磷酸化酶基因OsACO 1的表达诱导在一个时间依赖性的方式由热，而不是其他典型的非生物胁迫。为了分析水稻对热胁迫反应的转录调控机制，分离了OsACO 1启动子（P-OsACO 1），并对转基因水稻进行了鉴定。利用定性和定量分析，我们发现，GUS报告基因的表达响应于热在不同的组织和在不同的发展阶段时，驱动P-OsACO 1。产生一系列P-OsACO 1的5'末端缺失以描绘负责热诱导基因表达的区域。烟草叶片中的瞬时表达分析鉴定了-1386和-1065之间的322-bp最小区域，该区域对于P-OsACO 1的热诱导表达是必需的和足够的。我们筛选已知的热响应相关的顺式元件在这322 bp的区域，然而，与热诱导的基因表达相关的序列没有被确定在P-OsACO 1。因此，在该322-bp区域中进行截短和连续诱变分析。通过比较启动子片段及其衍生物的活性，我们的结果表明，热响应元件位于-1132和-1124之间的9-bp区域，该序列含有W-box基序。额外的定点突变分析消除了P-OsACO 1的热响应活性通过W-box元件，和电泳迁移率变动分析（EMSA）表明P-OsACO 1的结合因子在热胁迫水稻幼苗的核提取物中的W-box依赖的方式。我们的研究结果阐明了TCA响应非生物胁迫的一个关键组分的表达模式，并建立了水稻呼吸代谢基因响应热的转录调节的一个假定的调控途径。(C)2015爱思唯尔爱尔兰有限公司版权所有。
Respiratory metabolism is an important though poorly understood facet of plant adaptation to stress. Posttranslational modification of aconitase, a component of the tricarboxylic acid cycle (TCA), may be involved in stress tolerance. However, such stress-related transcriptional regulation and its mechanism remain unknown. In this study, we found that expression of the rice Aconitase gene OsACO1 is induced in a time-dependent manner by heat but not other typical abiotic stresses. To analyze the transcriptional regulation mechanism underlying the response to heat, the OsACO1 promoter (P-OsACO1) was isolated and characterized in transgenic rice. Using qualitative and quantitative analyses, we found that the expression of the GUS reporter gene responded to heat in different tissues and at different stages of development when driven by P-OsACO1. A series of 5' distal deletions of P-OsACO1 was generated to delineate the region responsible for heat-induced gene expression. Transient expression analyses in tobacco leaves identified a 322-bp minimal region between -1386 and -1065 as being essential and sufficient for heat-induced expression by P-OsACO1. We screened for known heat response-related cis-elements in this 322-bp region; however, sequences correlating with heat-induced gene expression were not identified in P-OsACO1. Therefore, truncations and successive mutagenesis analyses were performed in this 322-bp region. By comparing the activities of promoter fragments and their derivatives, our results indicated that the heat response element resided in a 9-bp region between -1132 and -1124, a sequence that contains a W-box motif. Additional site-directed mutagenesis analyses eliminated the heat response activity of P-OsACO1 via the W-box element, and an electrophoretic mobility shift assay (EMSA) indicated the binding of P-OsACO1 by factors in the nuclear extracts of heat-stressed rice seedlings in a W-box-dependent manner. Our results illustrate the expression pattern of a key component of the TCA response to abiotic stress and establish a putative regulatory pathway in the transcriptional modulation of rice respiratory metabolism genes in response to heat. (C) 2015 Elsevier Ireland Ltd. All rights reserved.