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：US06168945A

公开(公告)日：2001-01-02

申请号：US08404381

申请日：1995-03-14

申请人： John R. Sokatch ,  Pamela J. Sykes ,  K. T. Madhusudhan ,  Soo-Kyung Oh

发明人： John R. Sokatch ,  Pamela J. Sykes ,  K. T. Madhusudhan ,  Soo-Kyung Oh

IPC分类号： C12N1552

CPC分类号： C12N15/52 ,  C12N9/0008 ,  C12N15/78 ,  C12Y102/04004

摘要： DNA sequences coding for branched chain keto dehydrogenase, a promoter and branched chain keto dehydrogenase, and the promoter are disclosed. Also disclosed are recombinant vectors comprising one of the foregoing DNA sequences, transformed hosts comprising one of the foregoing recombinant vectors, and a method of making the polypeptide encoded by the DNA sequence.

摘要翻译： 公开了编码支链酮脱氢酶的DNA序列，启动子和支链酮脱氢酶，以及启动子。 还公开了包含前述DNA序列之一的重组载体，包含前述重组载体之一的转化宿主和由DNA序列编码的多肽的方法。