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首页> 外文期刊>Journal of Plant Physiology >Molecular cloning and characterization of a functional cDNA clone encoding isopentenyl diphosphate isomerase from Hevea brasiliensis
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Molecular cloning and characterization of a functional cDNA clone encoding isopentenyl diphosphate isomerase from Hevea brasiliensis

机译：巴西橡胶树编码异戊烯基二磷酸异构酶的功能性cDNA克隆的分子克隆和鉴定

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摘要

Plants produce many isoprenoid compounds of biological and/or economic importance. The first step in isoprenoid biosynthesis is the isomerization of isopentenyl diphosphate (IPP) to its electrophilic isomer, dimethylallyl diphosphate (DMAPP), by the enzyme IPP isomerase (EC 5.3.3.2). The following successive head-to-tail condensation reactions of the five-carbon intermediates lead to the synthesis of a variety of isoprenoids. Among these polyisoprenes, natural rubber is an important raw material with many industrial uses. In order to build our knowledge base of rubber biosynthesis, we cloned and characterized two cDNA clones encoding the IPP isomerase from the latex of Hevea brasiliensis, where a large quantity of natural rubber (cis-1,4-polyisoprene) is produced. The two clones differ only in the untranslated regions, and have a continuous open reading frame encoding a pep- tide of 234 amino acids with a predicted molecular mass of 26.7kDa. The deduced protein is acidic with an isoelectric point of 4.7, and shows high sequence identity with other IPP isomerases. The recombinant protein expressed in Escherichia coil showed IPP isomerase activity. In vitro rubber biosynthesis assays using washed rubber particles deprived of initiating allylic diphosphates, revealed that the recombinant IPP isomerase is functional in catalyzing the conversion of IPP to DMAPP, a key activation step of the basic five-carbon isoprene unit in isoprenoid biosynthesis. Southern analysis indicated that the IPP isomerase is encoded by two genes in Hevea rubber tree. Analyses of RNA extracted from extruded latex of the trees, wounded with nails, showed that wounding did not change the transcript level of IPP isomerase
机译：植物产生许多具有生物学和/或经济重要性的类异戊二烯化合物。异戊二烯生物合成的第一步是通过IPP异构酶（EC 5.3.3.2）将异戊烯基二磷酸（IPP）异构化为其亲电异构体二磷酸二甲基烯丙酯（DMAPP）。随后的五碳中间体从头到尾的缩合反应导致了各种类异戊二烯的合成。在这些聚异戊二烯中，天然橡胶是具有许多工业用途的重要原料。为了建立我们的橡胶生物合成知识基础，我们从巴西橡胶树胶乳中克隆并鉴定了两个编码IPP异构酶的cDNA克隆，并在其中生产了大量的天然橡胶（顺式1,4-聚异戊二烯）。这两个克隆仅在非翻译区有所不同，并具有一个连续的开放阅读框，其编码234个氨基酸的肽，预测分子量为26.7kDa。推导的蛋白质呈酸性，等电点为4.7，与其他IPP异构酶具有高度的序列同一性。在大肠杆菌中表达的重组蛋白具有IPP异构酶活性。使用不含起始烯丙基二磷酸酯的洗涤橡胶颗粒进行的体外橡胶生物合成测定显示，重组IPP异构酶在催化IPP向DMAPP的转化中起作用，这是类异戊二烯生物合成中基本五碳异戊二烯单元的关键活化步骤。 Southern分析表明，IPP异构酶由橡胶树中的两个基因编码。从树木的挤压乳胶中提取的，用钉子受伤的RNA的分析表明，受伤并没有改变IPP异构酶的转录水平

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