cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis)
cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis)
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DOI：
10.1006/abbi.1999.1332
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发表时间：
1999-08-15
影响因子：
3.9
通讯作者：
Croteau, R
中科院分区：
文献类型：
--
作者：
Bohlmann, J;Phillips, M;Croteau, R
大冷杉（Abies grandis）是研究针叶树防御性油树脂形成的生物化学、分子遗传学和调控的一个有用的模型系统，该过程涉及树脂（沥青）在专门的分泌结构中的组成性积累和单萜、倍半萜（松节油）和二萜树脂酸（松香）在损伤部位由非特化细胞诱导的生物合成。一个基于相似性的克隆策略，采用引物设计的保守区域的现有的单萜脱氢酶，并预计扩增1000 bp的片段，出乎意料地产生了300 bp的片段与序列联想到萜类化合物合酶。利用这种扩增子作为杂交探针提供了四个新的，全长的cDNA物种从受伤的冷杉茎cDNA文库，似乎编码四个不同的单萜脱氢酶。在大肠杆菌中表达，随后用香叶基二磷酸（C-10）、法呢基二磷酸（C-15）和香叶基香叶基二磷酸（C-20）进行酶活性测定，并通过手性相气相色谱和质谱分析萜烯产物，证实这些序列编码四种新的单萜脱氢酶，包括（-）-藜芦烯合酶、（-）-β-水芹烯合酶、萜品油烯合酶、以及产生（-）-柠檬烯和（-）-α-蒎烯的酶。推导的氨基酸序列表明这些酶的长度为618至637个残基（71至73 kDa），并被翻译为带有50-60个残基的氨基末端质体靶向序列的前蛋白。cDNA截短删除转运肽允许这些酶的“假成熟”形式的功能表达，其作为截短的结果在产物结果上没有表现出变化。序列比较显示，这些新的单萜脱氢酶从大冷杉Tpsd基因亚家族的成员，并类似于倍半萜（C-15）脱氢酶和二萜（C-20）脱氢酶从针叶树更密切的机制相关的单萜脱氢酶从被子植物物种。一个几乎完整的一套组成型和诱导型单萜脱氢酶从大冷杉（现在编号为7）的可用性将允许分子解剖的树脂为基础的防御反应，在这针叶树物种，和详细研究的结构-功能之间的关系，这个大的和不同的催化剂家族，所有这些都利用相同的立体化学在耦合的异构化-环化反应。(C)北京：科学出版社.
Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C-10), farnesyl diphosphate (C-15) and geranylgeranyl diphosphate (C-20), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (-)-camphene synthase, (-)-beta-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (-)-limonene and (-)-alpha-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50-60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the "pseudomature" forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C-15) synthases and diterpene (C-20) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure-function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization-cyclization reaction. (C) 1999 Academic Press.