cDNA cloning, substrate specificity and expression study of tobacco caffeoyl-CoA 3-O-methyltransferase, a lignin biosynthetic enzyme
cDNA cloning, substrate specificity and expression study of tobacco caffeoyl-CoA 3-O-methyltransferase, a lignin biosynthetic enzyme
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DOI：
10.1023/a:1005969825070
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发表时间：
1998-02
影响因子：
5.1
通讯作者：
F. Martz;S. Maury;G. Pinçon;M. Legrand
中科院分区：
文献类型：
--
作者：
F. Martz;S. Maury;G. Pinçon;M. Legrand
使用异源 DNA 探针从 TMV 感染的烟叶中提取的 RNA 中分离出四个咖啡酰辅酶 A 3-O-甲基转移酶 (CCoAOMT) cDNA 克隆。这些 cDNA 的核苷酸序列有 84-93% 的一致性，表明它们是四个密切相关的基因的产物。 CCoAOMT cDNA 与数据库序列的比较和 Southern 印迹分析表明它们是由烟草的新 CCoAOMT 家族编码的。通过 RNA 印迹分析研究了该基因家族在烟草组织中的总体表达。利用每个克隆的 cDNA 中存在的特定限制性位点，通过 RT-PCR 结合 PCR 产物的 RFLP 分析来研究每个单独基因的表达。 CCoAOMT 基因家族的两个成员似乎在各种植物器官和组织中组成型表达，而其他两个成员在烟草花叶病毒 (TMV) 感染或叶子的诱导子处理后优先在花器官中表达。纯化细菌中表达的 CCoAOMT 酶蛋白，并显示其对咖啡酰辅酶 A 和 5-羟基阿魏酰辅酶 A 酯具有特异性，并且对游离咖啡酸和 5-羟基阿魏酸没有活性。发现烟草茎发育过程中 CCoAOMT 转录本积累的模式与 COMT I 基因的积累模式密切相关，COMT I 基因已被证明专门参与木质素生物合成。此外，转基因烟草中 COMT I 基因表达的抑制也被证明会降低 CCoAOMT 基因表达，特别是在大多数木质化组织中。因此，烟草 CCoAOMT 的表达模式和底物特异性在木质素生物合成中维持优先作用。
Four caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) cDNA clones were isolated from RNA extracted from TMV-infected tobacco leaves using an heterologous DNA probe. The cDNAs were 84–93% identical in their nucleotide sequences, indicating that they are the products of four closely related genes. A comparison of the CCoAOMT cDNAs with database sequences and Southern blot analysis indicated that they are encoded by a new CCoAOMT family of tobacco. Overall expression of this gene family in tobacco tissues was investigated by RNA blot analysis. The expression of each individual gene was studied by RT-PCR coupled with RFLP analysis of PCR products, taking advantage of the presence of specific restriction sites in each cloned cDNA. Two members of the CCoAOMT gene family appeared to be constitutively expressed in various plant organs and tissues whereas the two others were preferentially expressed in flower organs, after tobacco mosaic virus (TMV) infection or elicitor treatment of leaves. The CCoAOMT enzymatic protein expressed in bacteria was purified and shown to be specific for the caffeoyl-CoA and 5-hydroxyferuloyl-CoA esters and to have no activity against free caffeic acid and 5-hydroxyferulic acid. The pattern of CCoAOMT transcript accumulation during development of tobacco stem was found closely related to that of COMT I genes which have been shown to be specifically involved in lignin biosynthesis. Moreover, the inhibition of COMT I gene expression in transgenic tobacco was also shown to decrease CCoAOMT gene expression, particularly in the most lignified tissues. Thus, the expression pattern and the substrate specificity of tobacco CCoAOMT sustain a preferential role in lignin biosynthesis.