MOLECULAR-CLONING AND EXPRESSION OF A NEW CLASS OF ORTHO-DIPHENOL-O-METHYLTRANSFERASES INDUCED IN TOBACCO (NICOTIANA-TABACUM-L) LEAVES BY INFECTION OR ELICITOR TREATMENT
MOLECULAR-CLONING AND EXPRESSION OF A NEW CLASS OF ORTHO-DIPHENOL-O-METHYLTRANSFERASES INDUCED IN TOBACCO (NICOTIANA-TABACUM-L) LEAVES BY INFECTION OR ELICITOR TREATMENT
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DOI：
10.1104/pp.103.2.509
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发表时间：
1993-10-01
期刊：
影响因子：
7.4
通讯作者：
LEGRAND, M
中科院分区：
文献类型：
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作者：
PELLEGRINI, L;GEOFFROY, P;LEGRAND, M
烟草（Nicotiana tabacum L.）cv Samsun NN）中，三种不同的酶解释了邻二酚-O-甲基转移酶（OMT）活性。OMT I是健康叶片的主要酶，而OMT II和III酶在对烟草花叶病毒（TMV）的过敏反应中优先被诱导。利用抗OMT Ⅲ的抗血清，我们通过免疫筛选从TMV感染的烟草叶片的mRNA制备的表达文库，分离了部分OMT Ⅲ cDNA克隆。使用该OMT III克隆作为探针，我们分离出具有推断的氨基酸序列的全长克隆，所述氨基酸序列包含通过Edman降解纯化的蛋白质II和III获得的所有序列。因此，烟草的OMT II和III很可能由相同的基因编码，并产生于不同的翻译后修饰。序列分析表明，该OMT克隆代表了一类新的OMT酶（II类），与以前从其他双子叶植物中克隆的OMT具有低水平的相似性（53-58%）。Southern杂交分析表明，烟草基因组中存在一个小家族的II类OMT基因，该基因遗传自与欧洲烟草和毛状烟草相关的祖先。RNA印迹分析表明，与I类OMT基因不同，II类OMT基因在烟草的木质部组织中不以高组成型水平表达。发现II类OMT转录物在感染TMV或用megaspermin（一种来自Phytophthora megasperma的蛋白质激发子）处理的烟叶中积累，但在用水杨酸（一种已知可触发许多防御基因的分子）处理的烟叶中不积累。在TMV感染或激发子处理的组织中，儿茶酚甲基化活性的显着增加伴随着II类OMT基因产物的积累。
In tobacco (Nicotiana tabacum L. cv Samsun NN), three distinct enzymes account for ortho-diphenol-O-methyltransferase (OMT) activity. OMT I is the major enzyme of healthy leaves, whereas enzymes OMT II and III are preferentially induced during the hypersensitive reaction to tobacco mosaic virus (TMV). Using an anti-OMT III antiserum, we isolated a partial OMT III cDNA clone by immunoscreening an expression library made from mRNA of TMV-infected tobacco leaves. Using this OMT III clone as a probe, we isolated a full-length clone with a deduced amino acid sequence encompassing all of the sequences obtained by Edman degradation of both purified proteins II and III. Thus, OMT II and III of tobacco are likely to be encoded by the same genes and to arise from different posttranslational modifications. Sequence analysis showed that this OMT clone represents a new class of OMT enzymes (class II) with a low level of similarity (53-58%) to OMTs cloned previously from other dicotyledonous plants. Southern analysis indicated that a small family of class II OMT genes inherited from ancestors related to Nicotiana sylvestris and Nicotiana tomentosiformis occurs in the tobacco genome. RNA blot analysis demonstrated that class II OMT genes, unlike class I OMT genes, are not expressed at a high constitutive level in lignified tissues of tobacco. Class II OMT transcripts were found to accumulate in tobacco leaves infected with TMV or treated with megaspermin, a proteinaceous elicitor from Phytophthora megasperma, but not in leaves treated with salicylic acid, a molecule known to trigger many defense genes. In TMV-infected or elicitor-treated tissues, a marked increase in catechol-methylating activity accompanied the accumulation of class II OMT gene products.