Cao Songxiao, Chen Hao, Zhang Chong, Tang Yufan, Liu Jieying, Qi Hongyan
Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, College of Horticulture, Shenyang Agricultural University, Shenyang, Liaoning, China.
PLoS One. 2016 Apr 21;11(4):e0153801. doi: 10.1371/journal.pone.0153801. eCollection 2016.
Lipoxygenases (LOXs) are a class of non-heme iron-containing dioxygenases that catalyse oxidation of polyunsaturated fatty acids to produce hydroperoxidation that are in turn converted to oxylipins. Although multiple isoforms of LOXs have been detected in several plants, LOXs in oriental melon have not attracted much attention. Two full-length LOX cDNA clones, CmLOX10 and CmLOX13 which have been isolated from oriental melon (Cucumis melo var. makuwa Makino) cultivar "Yumeiren", encode 902 and 906 amino acids, respectively. Bioinformatics analysis showed that CmLOX10 and CmLOX13 included all of the typical LOX domains and shared 58.11% identity at the amino acid level with each other. The phylogenetic analysis revealed that CmLOX10 and CmLOX13 were members of the type 2 13-LOX subgroup which are known to be involved in biotic and abiotic stress. Heterologous expression of the full-length CmLOX10 and truncated CmLOX13 in Escherichia coli revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the lipoxygenase activities. The purified CmLOX10 and CmLOX13 recombinant enzymes exhibited maximum activity at different temperature and pH and both had higher affinity for linoleic acid than linolenic acid. Chromatogram analysis of reaction products from the CmLOX10 and CmLOX13 enzyme reaction revealed that both enzymes produced 13S-hydroperoxides when linoleic acid was used as substrate. Furthermore, the subcellular localization analysis by transient expression of the two LOX fusion proteins in tobacco leaves showed that CmLOX10 and CmLOX13 proteins were located in plasma membrane and chloroplasts respectively. We propose that the two lipoxygenases may play different functions in oriental melon during plant growth and development.
脂氧合酶（LOXs）是一类含非血红素铁的双加氧酶，可催化多不饱和脂肪酸氧化生成氢过氧化物，进而转化为氧脂素。尽管在多种植物中已检测到多种LOX同工型，但甜瓜中的脂氧合酶尚未受到太多关注。从甜瓜（Cucumis melo var. makuwa Makino）品种“玉美人”中分离出两个全长LOX cDNA克隆，即CmLOX10和CmLOX13，它们分别编码902和906个氨基酸。生物信息学分析表明，CmLOX10和CmLOX13包含所有典型的LOX结构域，在氨基酸水平上彼此的同源性为58.11%。系统发育分析表明，CmLOX10和CmLOX13是2型13-LOX亚组的成员，已知该亚组参与生物和非生物胁迫。CmLOX10全长和截短的CmLOX13在大肠杆菌中的异源表达表明，编码的外源蛋白与预测的分子量一致，并具有脂氧合酶活性。纯化的CmLOX10和CmLOX13重组酶在不同温度和pH下表现出最大活性，并且两者对亚油酸的亲和力均高于亚麻酸。对CmLOX10和CmLOX13酶反应产物的色谱分析表明，当以亚油酸为底物时，两种酶均产生13S-氢过氧化物。此外，通过在烟草叶片中瞬时表达两种LOX融合蛋白进行亚细胞定位分析表明，CmLOX10和CmLOX13蛋白分别位于质膜和叶绿体中。我们认为这两种脂氧合酶可能在甜瓜的生长发育过程中发挥不同的功能。
