Cloning And Functional Analysis Of Terpene Biosynthesis Related Genes In Ligularia Fischeri

The genus Ligularia Cass is one of the large genera in Compositae- Senecioneae-Tussilagininae. In subtrib. Tussilaginac, Ligularia is closely related to, but more advanced than the genus Farfrrgizem Lindl. It includes six sections, 11 series and 129 species. All the taxa are distributed in Asia with only two species extending to Europe. There are 119 speciesin E. Asia, comprising 96 % of the world total. The triterpenoid glycosides are major chemical constituents of the genus Ligularia Cass., in addition that monoterpenes, sesquiterpenrs, diterpenes, steroids, coumarins, peptides are also isolated. Among of hem, some compounds possessed activities of antitumor, antibiotic, antiinflammation.3-hydroxy-3-methylglutaryl CoA reductase cDNA genes have been successfully isolated from Ligularia fischeri and named as HMGR with 2031 bp (GenBank accession number: DQ916106), which encodes a predicted polypeptide of 579 amino acids with 62.48kDa and pI 7.63 and HMGR2 with 1153 bp (GenBank accession number: EF133501), which encodes a predicted polypeptide of 343 amino acids with 36.33kDa and pI 8.44.The characteristics of HMGR and HMGR2 and their deducted amino acids were analyzed by the tools of bioinformatics in the following aspects: the composition of nuclei acid sequences and amino acid sequences, signal peptide, transmembrane topological structure, hydrophobicity or hydrophilicity, secondary structure of protein, molecular phylogenetic evolution and so on. The result s are as follows: the full-length gene of HMGR contains an opening readingframe, 5′and 3′-untranstrated regions; HMGR and HMGR2 are hydrophilic and transmembrane proteins which lack of signal peptide; the amino acid sequences of HMGRs include two functional HMG-CoA binding motifs and two functional NADPH binding motifs; the main motif of predicted secondary structure of HMGRs are alpha helix and random coil , beta turn and extended strand are spreaded in the whole secondary structure of protein.Southern blot analysis performed using a HMGR specific fragment as probe showed that two copies of HMGR gene in Ligularia fischeri. The RNA gel blot analysis showed a different HMGR expression patterns among the different organs at mRNA level with abundant amount in leaves. The determination of Shionone in roots and stems of Ligularia fischeri by HPLC showed rather high content of Shionone in leaves. To further investigate the function of HMGR, the overexpression of HMGR in Ligularia fischeri were alao studied by gene transformation via agrobacterium. PCR, Southern and Northern analyses of transgenic plants showed that the HMGR gene was integrated into the Ligularia fischeri genome and expressed at the transcriptional level. HPLC analysis showed that the Shionone content in transgenic roots and stems of Ligularia fischeri were increased 16.67% and 12.25% as compared with the control. It was suggested that overexpression of the HMGR gene could increase the content of Shionone and indicated that HMGR may play an important role in biosynthesis of Shionone.The cloning and characterizations of the above two genes would offer the theoretical basis to further investigate metabolic pathway of isoprenoid biosynthesis in plants.