Molecular Cloning And Characterization Of Important Genes Involved In The Biosynthetic Pathways Of Flavonoids And Terpenoids From Ginkgo Biloba L.

Ginkgo boliba L. is often called "living fossil" in plant kingdom, and its leaf extract contains many active ingredients for human health, among which ginkgo flavonoids and terpenoids are two of the most important ones. However, the overall biosynthetic pathways of flavonoids and terpenoids in G biloba are unclear at molecular genetic level. In order to deepen the research in this area, and to lay a groundwork for increasing contents of ginkgo flavonoids and terpenoids by using bioengineering in the near future, cloning and characterization of six key genes involved in the biosynthetic pathway of flavonoids and terpenoids, are presented in this text for the first time. Among them, four genes encoding chalcone synthase, flavanone 3-hydroxylase, anthocyanidin reductase and flavonol synthase participate in flavonoids biosynthesis, and other two genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase and mevalonate 5-disphosphate decarboxylase involve in MVA pathway in terpenoids biosynthesis.Chalcone synthase (CHS) catalyzes the first committed step in flavonoids biosynthesis. The full-length cDNA of CHS gene from G biloba (designated as GbCHS) and its genomic DNA sequence were obtained by using RACE and PCR. The comparison between cDNA and genomic DNA sequences revealed that GbCHS was composed of two exons and one intron, one of the typical features of CHS genes. GbCHS was found to have extensive homology with other plant CHS proteins via multiple alignments. The active sites of the CoA binding, coumaroyl pocket and cyclization pocket in putative GbCHS protein were the same as in that of Medicago sativa, the crystallogram and function of which has been elucidated. Homology-basedmolecular modeling revealed that the 3D model of GbCHS strongly resembled that of M. sativa (MsCHS2). Phylogenetic tree analysis revealed that GbCHS had closer relationship with CHSs from gymnosperm plant species than from other plant species. Southern blot analysis indicated that GbCHS belonged to a multigene family. RT-PCR analyses revealed that GbCHS expressed differentially in the root, stem and leaf, and the expression was induced by UV-B and wounding treatments. The GbCHS protein was successfully expressed in E. coli with the molecular weight being similar to that of the predicted by bioinformatics. Western blot analysis indicated that the rGbCHS containing His-tag had specific immune activity with anti-His antibodies. The plant expression vector pl304-GbCHS containing GbCHS was also constructed for plant transformation.Flavanone 3-hydroxylase (F3H) activity is necessary for the biosynthesis of flavonoids. The full-length cDNA and genomic DNA sequences of F3H gene were isolated from G biloba (designated as GbF3H) using RACE and PCR. The comparison between cDNA and genomic DNA sequences revealed that GbF3H was composed of three exons and two introns. The deduced GbF3H protein showed high identities to other plant F3Hs via multiple alignments. The conserved amino acids ligating ferrous iron and residues participating in 2-oxoglutarate binding (R-X-S) were identified in GbF3H at the similar positions like other F3Hs. Homology-based structure modeling showed that GbF3H model had a distorted jelly roll in the enzyme core which consisted of P-sheet, a typical structure shared by all 2-oxoglutarate-dependent dioxygenases including F3Hs. Phylogenetic tree analysis revealed that GbF3H shared the same ancestor in evolution with other F3Hs. Southern blot analysis indicated that GbF3H belonged to a multi-gene family. RT-PCR analysis revealed that GbF3H gene expressed in stem and leaf, with the highest expression in leaf. The GbF3H protein was successfully expressed in E. coli with the molecular weight being similar to that of the predicted by bioinformatics. The rGbF3H will be used for in vitro functional identification in the future.Flavonol synthase (FLS) directs the synthesis of flavonols, and flavonols and their glucosides are major ingredients of G biloba leaf extract. The full-length cDNA and genomic DNA sequences of FLS gene were isolated from G biloba using RACE and PCR. The comparison between cDNA and genomic DNA sequences revealed that GbFLS was composed of three exons and two introns. The 5' flanking region of GbFLS was isolated by genome walking technique, and some main cw-actingelements including TATA box and stress-responsiveness elements were predicted and analyzed. Multiple alignment analysis showed that GbFLS had identity to those of other plant species. Homology-based structure modeling showed that GbFLS 3D model had a distorted jelly roll in the enzyme core which consisted of P-sheet, a typical structure shared by all 2-oxoglutarate-dependent dioxygenases including FLSs. Phylogenetic tree analysis revealed that GbFLS shared the same ancestor with other FLSs. Southern blot analysis indicated that GbFLS gene belonged to a multigene family. RT-PCR analysis showed that GbFLS expressed in root, stem and leaf at different levels and the expression of GbFLS were induced by wounding, cold and UV-B treatments. The GbFLS protein was successfully expressed in E. coli with the molecular weight being similar to that of the predicted by bioinformatics. Western blot analysis indicated that the rGbFLS containing His-tag had specific immune activity with anti-His antibodies. The plant expression vector pl304-GbFLS containing GbFLS was also constructed for plant transformation.Anthocyanidin reductase (ANR) is an important enzyme involved in the biosynthesis of 2,3-frwisi-flavan-3-ol — the monomer of condensed tannins in plants. The full-length cDNA and its genomic DNA sequence of ANR gene from G biloba (designated as GbANR) were isolated using RACE and PCR. The comparison between cDNA and genomic DNA sequences revealed that GbANR had five exons and four introns. The deduced GbANR amino acid sequence had high identity to other plant ANRs via multiple alignments. Phylogenetic tree analysis revealed that GbANR shared the same ancestor with other known ANR proteins. Southern blot analysis indicated that GbANR belonged to a small multigene family. RT-PCR analysis indicated that GbANR expressed in stem and leaf. GbANR was constructed into E. coli expression vector and the recombinant plasmid harboring GbANR gene will be used for the further protein expression and purification.The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the first committed step in the MVA pathway for biosynthesis of terpenoids. The full-length cDNA and its genomic DNA sequence of HMGR gene from G biloba (designated as GbHMGR) were isolated using RACE and PCR. The comparison between cDNA and genomic DNA sequences revealed that GbHMGR had four exons and three introns. The deduced GbHMGR amino acid sequence had high identity to other plant HMGRs via multiple alignments. Phylogenetic tree analysis revealed that GbHMGR shared the same ancestor with other known HMGR proteins from plant...