公开(公告)号：US06316695B1

公开(公告)日：2001-11-13

申请号：US09296754

申请日：1999-04-22

申请人： Kyung-Han Han ,  Hun-Seung Kang ,  Soo-Kyung Oh ,  Dong-Ho Shin ,  Jae-Mo Yang

发明人： Kyung-Han Han ,  Hun-Seung Kang ,  Soo-Kyung Oh ,  Dong-Ho Shin ,  Jae-Mo Yang

IPC分类号： C12N1582

CPC分类号： C08F136/08 ,  C12N9/90 ,  C12N15/8243 ,  C12P5/007 ,  Y10S435/814

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.

摘要翻译： 本发明克隆了编码异戊烯基二磷酸（以下称为“IPP”）异构酶的cDNA克隆（EC 5.3.3.2），其从巴西三叶胶乳的cDNA文库中克隆。 克隆具有编码234个氨基酸的肽的连续开放阅读框，其预测分子量为26.7kDa。 推测的蛋白质为酸性，等电点为4.7，与其他IPP异构酶显示高序列同一性。 在大肠杆菌中表达的重组蛋白显示IPP异构酶活性。 在反应混合物中加入IPP异构酶，使用剥夺了引发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转化为DMAPP时具有催化活性，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。 南方分析表明IPP异构酶由双叶橡胶树中的两个基因编码。 在Northern印迹分析中，从叶组织中检测到两种不同大小的转录物（1.2和0.6kb），而从胶乳中仅检测到一个杂交带（1.0kb）。 用指甲树的挤出乳胶和叶组织提取的RNA的分析显示，伤口没有改变IPP异构酶的转录水平。

公开(公告)号：US06235860B1

公开(公告)日：2001-05-22

申请号：US09322639

申请日：1999-05-28

申请人： Hun-Seung Kang ,  Kyung-Hwan Han

发明人： Hun-Seung Kang ,  Kyung-Hwan Han

IPC分类号： C08G8300

CPC分类号： C08C1/00 ,  Y10S528/931

摘要： Natural rubber products are made from fig tree (Ficus carica) by homogenizing the plants in an acetone/benzene and separating the rubber-containing phase by centrifugation. F. carica contains about 4% (w/v) natural rubber in latex, and 0.3, 0.1 and 0.1 % in bark, leaf and fruit, respectively. Molecular mass of the natural rubber from F. carica is about 128 kD. The latex serum of F. carica contains only a small numbers of proteins, with a 48 kD and a 25 kD protein as major proteins. Similar to Hevea brasiliensis and Parthenium argentatum Gray, proteins participating in rubber biosynthesis of F. carica are tightly associated with the rubber particles. Natural rubber is made using the latex serum and the proteins in latex, leaf, bark, and fruit of F. carica.

摘要翻译： 天然橡胶制品由无花果树（Ficus carica）制成，通过在丙酮/苯中均匀化植物，并通过离心分离含橡胶的相。 car ica分别含有胶乳中约4％（w / v）的天然橡胶，分别含有0.3,0.1和0.1％的树皮，叶子和水果。 来自樟树的天然橡胶的分子质量约为128kD。 F.carica的乳胶血清只含有少量蛋白质，以48kD和25kD蛋白为主要蛋白质。 类似于三叶草（Brassiliensis）和金缕梅（Gray），参与橡胶生物合成的蛋白质与橡胶颗粒紧密相关。 天然橡胶使用胶乳血清和乳胶，叶，树皮和果实中的蛋白质制成。