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Isopentenyl diphosphate isomerase from Hevea brasiliensis and rubber producing method using the same

机译:巴西橡胶树的异戊烯基二磷酸异构酶及其橡胶生产方法

摘要

The present invention cloned a cDNA clone encoding isopentenyl diphosphate (hereafter “IPP”) isomerase (EC 5.3.3.2) from a cDNA library of Hevea brasiliensis latex. The clone has a continuous open reading frame encoding a peptide of 234 amino acids with a predicted molecular mass of 26.7 kDa. 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 coli showed IPP isomerase activity. In vitro rubber biosynthesis assays using washed rubber particle (WRP) deprived of initiating allylic diphosphates were performed with the addition of IPP isomerase in the reaction mixture. Results revealed that the recombinant IPP isomerase is catalytically active in catalyzing the conversion of IPP to DMAPP, a key activation step of the basic five-carbon isoprene unit in rubber biosynthesis. Southern analysis indicated that the IPP isomerase is encoded by two genes in Hevea rubber tree. In Northern blot analysis, two different sizes of transcripts (1.2 and 0.6 kb) were detected from leaf tissues while only one hybridizing band (1.0 kb) was detected from latex. Analyses of RNA extracted from extruded latex and leaf tissues of the trees wounded with nails showed that wounding did not change the transcript level of IPP isomerase.
机译:本发明从巴西橡胶树(Hevea brasiliensis)乳胶的cDNA文库中克隆了编码异戊烯基二磷酸异磷酸酯酶(以下称为“ IPP”)异构酶(EC 5.3.3.2)的cDNA克隆。该克隆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26.7 kDa。推导的蛋白质呈酸性,等电点为4.7,并且与其他IPP异构酶具有高度的序列同一性。大肠杆菌中表达的重组蛋白具有IPP异构酶活性。在反应混合物中添加IPP异构酶后,使用缺少起始烯丙基二磷酸酯的洗涤橡胶颗粒(WRP)进行体外橡胶生物合成测定。结果表明,重组IPP异构酶在催化IPP向DMAPP的转化中具有催化活性,这是橡胶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。在Northern印迹分析中,从叶片组织中检测到两种不同大小的转录本(1.2和0.6 kb),而从乳胶中仅检测到一个杂交带(1.0 kb)。从挤压指甲的树木的乳胶和叶子组织中提取的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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