Abstract:

2C methyl D erythritol 2,4 cyclodiphosphate synthase (IspF) is the fifth enzyme involved the MEP pathway.The gene was cloned from Ginkgo biloba by using RACE technology, and was designated as GbIspF (GenBank accession No.: EF062579). The fulllength cDNA of GbIspFis 897 bp containing a 720 bp open reading frame (ORF) encoding a 239aminoacid polypeptide with a 59aminoacid plastidial transit peptide at its Ntermius. The multiple alignment analysis showed GbIspF was homologous with IspFs derived from other plant species. Semiquantitative RTPCR was carried out to investigate the tissue expression profile of GbIspF in different tissues including roots, stems, leaves and seeds. The results showed that GbIspF expression could be detected in all the tissues but at different levels. The highest expression level was found in leaves while the lowest expression level was found in roots. Functional complementation assay indicated that GbIspFcould promote the βcarotene accumulation in engineered XL1Blue haboring pTrcGbIspF and pACBETA, and as a result the engineered bacteria showed the brightly orange color given by βcarotene. This suggested that GbIspF had the typical function of known IspF genes.

Key words: Ginkgo biloba, IspF, cloning, bioinformatic analysis, functional complementation