CLONING AND HETEROLOGOUS EXPRESSION OF A 2ND (-DELTA-CADINENE SYNTHASE FROM GOSSYPIUM-ARBOREUM())

Screening of a Gossypium arboreum L. cv. Nanking cDNA library resulted in the identification and cloning of a second (+)-delta-cadinene synt hase. A probe for the screens was prepared by PCR using primers based on conserved sequences in farnesyl diphosphate cyclases and genomic DN A 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 homo logy 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 treatme nt of cotton cell suspension cultures with a partially purified elicit or preparation from the phytopathogenic fungus Verticillium dahliae. E xpression of CAD1-A mRNA was quantitated with reverse transcription PC R and showed that CAD1-A mRNA was maximally increased 8-fold, 6 h afte r addition of elicitor. Heterologous expression of this second cDNA pr oduced a 64 kD protein that catalyzed the cyclization of farnesyl diph osphate to (+)-delta-cadinene, the identical product produced by CAD-C 1. The steady-state kinetic parameters of CAD1-A were similar to CAD1- C, showing a K-m of 7 mM farnesyl diphosphate and k(cat) 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.