从烟草（Nicotiana tabacum）中克隆、鉴定和表达分析两种 12-氧代-植物二烯酸还原酶（NtOPR1 和 NtOPR2）。
Molecular cloning, characterization and expression analysis of two 12-oxophytodienoate reductases (NtOPR1 and NtOPR2) from Nicotiana tabacum.
机构信息
College of Agriculture and Life Sciences, Yunnan Urban Agricultural Engineering & Technological Research Center, Kunming University, Kunming, China.
The Research Center for Ornamental Plants, College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou, 510642, China.
出版信息
Mol Biol Rep. 2022 Jun;49(6):5379-5387. doi: 10.1007/s11033-022-07114-9. Epub 2022 Feb 11.
BACKGROUND
12-oxophytodienoic acid (OPDA) is a signaling molecule involved in defense and stress responses in plants. 12-oxophytodienoate reductase (OPR) is involved in the biosynthesis of jasmonic acid and trigger the conversion of OPDA into 3-oxo-2(2'[Z]-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0).
METHODS AND RESULTS
Sequence analysis revealed that Nicotiana tabacum 12-oxophytodienoate reductase 1 (OPR1) and OPR2 encoded polypeptides of 375 and 349 amino acids with molecular masses of 41.67 and 39.04 kilodaltons (kDa), respectively, while the deduced protein sequences of NtOPR1 and NtOPR2 showed high homology with other 12-oxophytodienoate reductases. BLAST (Basic local alignment search tool) analysis revealed that both NtOPRs belong to the family of Old Yellow Enzymes (OYE), and analysis of genomic DNA structure indicated that both genes include 5 exons and 4 introns. Phylogenetic analysis using MEGA X showed that NtOPR1 and NtOPR2 shared a close evolutionary relationship with Nicotiana attenuata 12-oxophytodienoate reductases. In silico analysis of subcellular localization indicated the probable locations of NtOPR1 and NtOPR2 to be the cytoplasm and the peroxisome, respectively. Tissue-specific expression assays via qRT-PCR revealed that NtOPR1 and NtOPR2 genes were highly expressed in Nicotiana tabacum roots, temperately expressed in leaves and flowers, while low expression was observed in stem tissue.
CONCLUSIONS
Presently, two 12-oxophytodienoate reductase genes (NtOPR1 and NtOPR2) were cloned and comprehensively characterized. Our findings provide comprehensive analyses that may guide future deep molecular studies of 12-oxophytodienoate reductases in Nicotiana tabacum.
背景
12-氧代-植物二烯酸（OPDA）是一种参与植物防御和应激反应的信号分子。12-氧代-植物二烯酸还原酶（OPR）参与茉莉酸的生物合成，并触发 OPDA 转化为 3-氧代-2（2'[Z]-戊烯基）-环戊烷-1-辛酸（OPC-8：0）。
方法和结果
序列分析表明，烟草 12-氧代-植物二烯酸还原酶 1（OPR1）和 OPR2 编码的多肽分别为 375 和 349 个氨基酸，分子量分别为 41.67 和 39.04 千道尔顿（kDa），而 NtOPR1 和 NtOPR2 的推导蛋白序列与其他 12-氧代-植物二烯酸还原酶具有高度同源性。BLAST（基本局部比对搜索工具）分析表明，两种 NtOPRs 都属于 Old Yellow Enzymes（OYE）家族，基因组 DNA 结构分析表明，这两个基因都包括 5 个外显子和 4 个内含子。使用 MEGA X 进行的系统发育分析表明，NtOPR1 和 NtOPR2 与烟草 12-氧代-植物二烯酸还原酶具有密切的进化关系。亚细胞定位的计算机分析表明，NtOPR1 和 NtOPR2 的可能位置分别为细胞质和过氧化物酶体。通过 qRT-PCR 进行的组织特异性表达分析表明，NtOPR1 和 NtOPR2 基因在烟草根中高度表达，在叶片和花朵中适度表达，而在茎组织中表达水平较低。
结论
目前，已经克隆并全面表征了两个 12-氧代-植物二烯酸还原酶基因（NtOPR1 和 NtOPR2）。我们的研究结果提供了全面的分析，可能为未来烟草 12-氧代-植物二烯酸还原酶的深入分子研究提供指导。
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