苹果丙二烯氧化物合酶MdAOS的克隆和表达分析

丙二烯氧化物合酶（allene oxide synthase,AOS）是茉莉酸合成途径中第一个特异性的酶。本研究从＂嘎啦＂苹果中克隆了一个AOS的基因序列,将其命名为MdAOS,并对其进行了结构和表达分析。MdAOS基因编码区共有1 605 bp碱基,编码531个氨基酸,等电点为4.92,分子量为134.9 kD。MdAOS属于细胞色素P450基因家族的CYP74A亚类,并具有典型的P450成员特征,其编码的蛋白包含4个保守区域,分别为：Heme-Binding结构域、Ⅰ-Helix区、ETLR基序和PEEF基序。MdAOS蛋白的N末端有典型叶绿体信号肽。同源性分析表明MdAOS与碧桃（Amygdalus persica var.persica f.duplex.）AOS基因的同源性很高,且聚类结果也与双子叶植物聚为一类。实时荧光定量PCR分析结果显示,MdAOS在苹果各组织中均有表达且在茎中表达量最高。MdAOS能响应茉莉酸、水杨酸、脱落酸和乙烯的诱导,并在机械伤处理时表达上调。

Cloning and Expression Analysis of an Allene Oxide Synthase Gene MdAOS from Malus domestica

Allene oxide synthase（AOS） is the first specific enzyme in jasmonic acid biosynthesis pathway. In the present study, an apple AOS gene was cloned from＂Gala＂apple（Malus domestica Borkh cv） and named as MdAOS. The gene structure and its expression in different tissues were analysed by using quantative real time PCR. Open reading frame of MdAOS from apple encompassed 1 605 bp encoding a polypeptide of 531 amino acids with calculated protein molecular mass of 134.95 kD. Bioinformatic analysis indicated that pI were 4.92. MdAOS belonged to CYP74A subgroup of P450 family and possessed typical feature of P450 family, such as Heme-Binding domain, Ⅰ-Helix motif, ETLR motif and PEEF motif. MdAOS protein contained a chloroplast signal peptide in N terminus. The MdAOS was highiy homologous to AOS of Amygdalus persica var. persica f. duplex. and they were clustered together with other AOS proteins of dicotyledonea. Quantitative real-time PCR analysis showed that MdAOS was expressed in all tissues of apple, with the highest expression level in stems. Moreover, MdAOS was significantly induced by the treatment of methyl jasmonate（JA）, salicylic acid（SA）, abscisic acid（ABA） and 1-aminocyclopropane-1-carboxylic acid（ACC）, as well as wounding treatment.