Rubber production process using isopentoxy enyl diphosphates isomerase from hevea brasiliensis

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.