Cloning and Heterologous Expression of a Second (+)-δ-Cadinene Synthase from Gossypium arboreum
| Autor: | Wang M, Xiao-Ya Chen, Peter Heinstein, Davisson Vj, Chen Y |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Recombinant Fusion Proteins
Molecular Sequence Data Pharmaceutical Science Molecular cloning Polymerase Chain Reaction Analytical Chemistry Open Reading Frames Complementary DNA Drug Discovery Gene expression Genomic library Amino Acid Sequence Cloning Molecular Isomerases Cells Cultured Gene Library Pharmacology Plants Medicinal Base Sequence ATP synthase biology cDNA library Organic Chemistry Blotting Northern Molecular biology Elicitor Molecular Weight Kinetics Complementary and alternative medicine biology.protein Molecular Medicine Electrophoresis Polyacrylamide Gel Mitosporic Fungi Heterologous expression |
| Zdroj: | Journal of Natural Products. 59:944-951 |
| ISSN: | 1520-6025 0163-3864 |
| Popis: | Screening of a Gossypium arboreum L. cv. Nanking cDNA library resulted in the identification and cloning of a second (+)-delta-cadinene synthase. A probe for the screens was prepared by PCR using primers based on conserved sequences in farnesyl diphosphate cyclases and genomic DNA as a template. This second cDNA clone encodes a protein that is 80% identical to the recently described (+)-delta-cadinene synthases CAD1-C1 and C14 from G. arboreum and maintains a significant degree of homology to the other known mono-, sesqui-, and diterpene synthases. As in the case of CAD1-C1 (+)-delta-cadinene synthase from cultured cotton cells, the synthesis of the second CAD1-A mRNA was induced by treatment of cotton cell suspension cultures with a partially purified elicitor preparation from the phytopathogenic fungus Verticillium dahliae. Expression of CAD1-A mRNA was quantitated with reverse transcription PCR and showed that CAD1-A mRNA was maximally increased 8-fold, 6 h after addition of elicitor. Heterologous expression of this second cDNA produced a 64 kD protein that catalyzed the cyclization of farnesyl diphosphate to (+)-delta-cadinene, the identical product produced by CAD1-C1. The steady-state kinetic parameters of CAD1-A were similar to CAD1-C, showing a Km of 7 mM farnesyl diphosphate and kcat of 0.039 s-1 at 30 degrees C. However, the optimal pH and Mg2+ concentration for CAD1-A activity were significantly higher than those observed for CAD1-C. |
| Databáze: | OpenAIRE |
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