| The flavonoids of Ginkgo biloba have many beneficial pharmaceutical properties for human health. The studies on increasing the content of flavonoids in G. biloba had been more and more popular and important. Until now, many methods had been carried out to increase the content of G. biloba flavonoids, and it was available method via physiological and biochemical to increase flavonoid content. In addition, it will be an effective method by genetic engineering to increase flavonoid content in the near future. However, the overall biosynthetic pathway of flavonoids in G. biloba is unclear at molecular genetic level. In order to deepen the research in this area, and to lay a groundwork for increasing the content of ginkgo flavonoids by using bioengineering, cloning and characterization of two key genes GbPAL and GbANS, which encoding phenylalanine ammonia-lyase and anthocyanidin synthase, involved in the biosynthetic pathway of flavonoids, are presented in this text for the first time. Moreover, to provide a new theory basis and practical reference for promoting the accumulation of flavonoids of ginkgo leaves, the effects of 5-aminolevulinic acid (ALA), a key precursor in the biosynthesis of porphyrins, in a low level (10 and 100 mg/l) on photosynthesis, flavonoids accumulation in ginkgo leaves were also first investigated in this research. The main contents and results in the present study are as following:(1) Molecular cloning, characterization and expression of phenylalanine mmonia-lyase gene from G. biloba.A full-length cDNA and genomic DNA of phenylalanine ammonia-lyase gene, which catalyzes the first step in the flavonoid biosynthetic pathway, were isolated from G. biloba for the first time (designated as GbPAL, GenBank Accession No. EU071050). The cDNA and genomic sequences of GbPAL were the same; in other words, this gene is intronless. The coding region of the gene was 2172 bp long, and its deduced protein consists of 724 amino acids with a predicted molecular mass of 79.1 kDa and a pI of 5.96. The deduced GbPAL protein showed high identities to other plant PALs. Southern hybridization analysis indicated that GbPAL belonged to a small multi-gene family. Tissue expression analysis by real-time PCR revealed that GbPAL constitutively expressed in all the tested tissues, especially highly in leaf and stem. GbPAL was also observed to be induced by a variety of stresses including UV-B, wounding, cold and salicylic acid. Temporal expression profiling analyses showed that the transcription levels of GbPAL were significantly correlated with flavonoid accumulation, suggesting that GbPAL might play a regulatory role in flavonoid biosynthesis in leaves of G. biloba at the transcriptional level.(2) Molecular cloning and function analysis of anthocyanidin synthase gene from G. biloba, and its expression in abiotic stress responses.Anthocyanidin synthase (ANS, leucoanthocyanidin oxygenase), a 2-oxoglutarate iron-dependent oxygenase, catalyzed the penultimate step in the biosynthesis of the anthocyanin class of flavonoids, from the colorless leucoanthocyanidins to the colored anthocyanidins. The full-length cDNA (EU600206) and genomic DNA (EU600205) sequences of ANS gene (designated as GbANS) were isolated from G. biloba for the first time. The full-length cDNA of GbANS contained a 1062-bp open reading frame (ORF) encoding a 354-amino-acid protein. The genomic DNA sequence analysis showed that GbANS gene had three exons and two introns. The 5′flanking region of GbANS was isolated by genome walking method, and some main cis-acting elements including TATA box and stress-responsiveness elements were predicted and analyzed. The deduced GbANS protein showed high identities to other plant ANSs. The conserved amino acids ligating ferrous iron (H-X-D) and residues participating in 2-oxoglutarate binding (R-X-S) were found in GbANS at the similar positions like other ANSs. 3D structure modeling showed that GbANS had a jellyroll motif in the enzyme core consisted ofβ-sheet, a typical structure shared by all 2-oxoglutarate-dependent dioxygenases including ANSs. Phylogenetic tree analysis revealed that GbANS shared the same ancestor with other ANSs. Southern blot analysis indicated that GbANS belonged to a multi-gene family. The expression analysis by real-time PCR showed that GbANS expressed in a tissue-specific manner in G. biloba, which were in good agreement with the pattern of anthocyanins accumulation in G. biloba. GbANS was also found to be up-regulated by all of the six tested abiotic stresses, namely UV-B, abscisic acid, sucrose, salicylic acid, cold and ethylene, consistent with the promoter region analysis of GbANS. The in vitro enzyme activity assay by HPLC indicated that recombinant GbANS protein could catalyze the formation the cyanidin from leucocyanidin and conversion of dihydroquercetin to quercetin, suggesting GbANS is a bifunctional enzyme within the anthocyanidin and flavonol biosynthetic pathway.(3) Effect of 5-aminolevulinic acid on flavonoids accumulation in the leaves of G. biloba. The effects of 5-aminolevulinic acid (ALA), a key precursor in the biosynthesis of porphyrins, in the low levels (10 and 100 mg/l) on photosynthesis, flavonoids accumulation and the activities of three enzymes involved in flavonoids biosynthesis in Ginkgo biloba leaves were investigated. The results showed that photosynthetic rates of leaves that treated with both concentrations of ALA, increased significantly at day 4 compared with that of control and remained so for 16 days. ALA at concentrations of 10 and 100 mg/l significantly increased the contents of chlorophyll and soluble sugar (P<0.05) at day 4 and these increments kept increase tendency until day 16, while the ratio of Chl a/b remained constant after ALA treatment. ALA-treatment enhanced the contents of total polyphenols, flavonoids and anthocyanins, as well as PAL, CHS and CHI activities from day 4 to 16. The effects on those determined targets mentioned above were all concentration-dependent, and 100mg/L ALA had greater effect than 10 mg/L ALA. These results suggestted that foliar treatment with the low concentration of ALA might provide a useful means of improving pharmacological properties in G. biloba leaves. |
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