Kiyosue T, Beetham J K, Pinot F, Hammock B D, Yamaguchi-Shinozaki K, Shinozaki K
Laboratory of Plant Molecular Biology, Institute of Physical and Chemical Research (RIKEN), Ibaraki, Japan.
Plant J. 1994 Aug;6(2):259-69. doi: 10.1046/j.1365-313x.1994.6020259.x.
A cDNA (1122 bp) was isolated from a cDNA library prepared from Arabidopsis thaliana L. that had been subjected to drought stress for 1 h. The sequencing of a genomic clone corresponding to the cDNA and S1 mapping analysis revealed that the cDNA lacked the first 6 bp from its translational start (ATG). The resulting open reading frame encodes a polypeptide of 321 amino acids, and the calculated molecular weight of this polypeptide is 36,423 Da. The deduced amino acid sequence shows a high degree of similarity to C terminal halves of those of soluble epoxide hydrolases (sEHs) of human, mouse and rat, 35.5%, 34.1% and 33.1%, respectively. The cDNA was expressed in Escherichia coli cells, and the expressed protein migrates at 40 kDa when analyzed by SDS-PAGE. The recombinant protein at 40 kDa is much smaller than the mammalian sEH (58 kDa) but has characteristics of activity and inhibition similar to the mammalian sEHs when assayed with the substrate trans-stilbene oxide and the inhibitors 4-fluorochalcone oxide (4FCO), (2R,3R)-3-(4-nitrophenyl) glycidol (RRNPG), and (2S,3S)-3-(4-nitrophenyl)glycidol (SSNPG), which indicates that the cDNA did encode a soluble epoxide hydrolase of A. thaliana (AtsEH). Drought stress, but not heat or cold stress, slightly increased the accumulation of the mRNAs for AtsEH. The level of AtsEH transcripts increased strongly after treatment with a plant hormone, auxin (2,4-dichlorophenoxyacetic acid, 2,4-D; naphthalene-acetic acid, NAA; and indole-3-acetic acid, IAA) in young, pre-bolting plants. Treatment with cytokinin (6-benzylaminopurine, BA), abscisic acid (ABA) or gibberellin (GA3) had no detectable effect on AtsEH transcript levels. The transcripts for AtsEH gene were detected in the aerial vegetative organs of bolting plants (i.e. stems and leaves), but not in roots, flowers and seeds. The possible function of AtsEH is discussed. A similar sEH cDNA has recently been characterized in potato (Stapleton et al., 1994).
从经干旱胁迫1小时的拟南芥制备的cDNA文库中分离出一个cDNA（1122 bp）。对与该cDNA对应的基因组克隆进行测序和S1图谱分析表明，该cDNA在其翻译起始位点（ATG）缺失前6个碱基对。所得的开放阅读框编码一个由321个氨基酸组成的多肽，该多肽的计算分子量为36423 Da。推导的氨基酸序列与人、小鼠和大鼠的可溶性环氧化物水解酶（sEHs）的C末端部分分别具有35.5%、34.1%和33.1%的高度相似性。该cDNA在大肠杆菌细胞中表达，经SDS-PAGE分析，表达的蛋白迁移率为40 kDa。40 kDa的重组蛋白比哺乳动物的sEH（58 kDa）小得多，但在用反式芪氧化物作为底物以及4-氟查耳酮氧化物（4FCO）、（2R,3R）-3-（4-硝基苯基）缩水甘油（RRNPG）和（2S,3S）-3-（4-硝基苯基）缩水甘油（SSNPG）作为抑制剂进行检测时，具有与哺乳动物sEHs相似的活性和抑制特性，这表明该cDNA确实编码了拟南芥的一种可溶性环氧化物水解酶（AtsEH）。干旱胁迫而非热胁迫或冷胁迫略微增加了AtsEH的mRNA积累。在用植物激素生长素（2,4-二氯苯氧乙酸，2,4-D；萘乙酸，NAA；以及吲哚-3-乙酸，IAA）处理幼嫩的抽薹前植株后，AtsEH转录本水平大幅增加。用细胞分裂素（6-苄基氨基嘌呤，BA）、脱落酸（ABA）或赤霉素（GA3）处理对AtsEH转录本水平没有可检测到的影响。在抽薹植株的地上营养器官（即茎和叶）中检测到了AtsEH基因的转录本，但在根、花和种子中未检测到。文中讨论了AtsEH的可能功能。最近在马铃薯中鉴定出了一个类似的sEH cDNA（Stapleton等人，1994年）。
