Molecular Cloning Important Genes Involved In The Accumulative Process For Flavonoids And Function Of Chalcone Synthase Gene Promoter From Ginkgo Biloba L.

In order to study biosynthetic mechanism of flavonoids in G.biloba and develop biotechnology of increasing the content of the flavonoids, several key genes such as GbCHI, GbF3'H, GbCCR, GbEPSPs and GbCHSp which are involved in the flavonoids accumulation process have been cloned and studied in this paper. The main results are as follows:(1) Molecular cloning, characterization and expression models of chalcone isomerase gene from Ginkgo biloba. A full-length cDNA and genomic DNA of chalcone isomerase gene were isolated from Ginkgo biloba L. by using PCR and RACE technologies. Compared with the cDNA sequence, the genomic sequence contain two intron and three extron. The coding region of the gene is 735 bp long, and its deduced protein consists of 244 amino acids with a predicted molecular mass of 26.29 kDa and a pI of 7.76. Protein sequence analysis reveals that GbCHI had closer relationship with TypeⅠCHIs. Phylogenetic tree analysis revealed that GbCHI does not belong to TypeⅠor typeⅡgroup. Southern blot analysis indicated that GbF3'H belonged to a multi-gene family. The results of prokaryotic expression showed that the product of recombinant GbCHI protein was in accordance with the anticipation. The vitro enzyme activity assay by HPLC indicated that recombinant GbCHI protein could catalyze the formation the (2S) naringenin from 6'-hydroxychalcone. The expression analysis by RT-PCR showed that GbCHI constitutively expressed in all the tested tissues, and had tissue specific manner in G.biloba. GbCHI was also found to be up-regulated by UV, ALA, ETH, ABA, CCC, and be down-regulated by GA. Correlation analysis between CHI activity and flavonoid accumulation during gingkgo leaf growth indicated that GbCHI might be the rate-limiting enzyme in the biosynthesis pathway of flavonoids in ginkgo leaves. Results of quantitative RT-PCR analysis showed that CHI activity correlated with the transcription level of change in CHI gene, suggesting CHI gene as the specific key gene regulating flavonoid accumulation in ginkgo.(2) Molecular cloning, characterization and expression models of Flavonoid 3'-hydroxylase gene from G.biloba. Flavonoid 3'-hydroxylase (GbF3'H), is a member of the P450 superfamily, which catalyzes monooxygenase reactions dependent upon NADPH and O2. In the flavonoid pathway F3'H hydroxylates the 3'-position of the B ring of naringenin and dihydrokaempferol to generate eriodictyol and dihydroquercetin, respectively which are important intermediates for biosynthesis of anthocyanins and proanthocyanidins, major coloration substances of flowers and seed coat. The full-length cDNA sequences of F3'H gene (designated as GbF3'H) were isolated from G.biloba for the first time. The full-length cDNA of GbF3'H contains a 1671 bp open reading frame (ORF) encoding a 556 amino acid protein. The 5'flanking region of GbF3'H 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 GbF3'H protein showed low identities to other plant F3'Hs, but had closer relationship with Cichorium intybus and shares 56.3% homology.3D structure modeling showed that GbF3'H bears all conserved motifs featured and have high similarity with P450s. Phylogenetic tree analysis revealed that the divergence time of GbF3'H from other F3'Hs is earlier. Southern blot analysis indicated that GbF3'H belonged to a multi-gene family. The expression analysis by RT-PCR showed that GbF3'H expressed in a tissue-specific manner in G.biloba, with the highest level in stamen and next in mature leaves. GbF3'H was also found to be up-regulated by the five tested abiotic stresses:UV-B,6-BA,SA,ABA and IAA, but non significant effect to wounding. Function analysis suggest that GbF3'H is a functional enzyme within the anthocyanidin and flavonol biosynthetic pathway.(3) Molecular cloning, characterization and expression models of EPSP synthase Gene From Ginkgo biloba L. The full-length cDNA sequences of EPSP synthase gene (designated as GbEPSPs) were isolated from G.biloba by using PCR and RACE technologies for the first time. The full-length cDNA of GbEPSPs is 1404bp and contains a 1035 bp open reading frame (ORF) encoding a 344 amino acid peptide sequence. Protein sequence analysis reveals that GbEPSPs had close relationship with other EPSPs and shares 81%-84% homology. Phylogenetic tree analysis showed that GbEPSPs as a gymnosperm and other angiosperm EPSPs are clustered to one monophyletic group, but the divergence time is early. RT-PCR analysis showed that GbEPSPs expressed in leaves, stems, roots and fruits, and had the highest expression in leaves and fruits, the next in stems, the least in roots. The expression of GbEPSPs could be induced by glyphosate and UV-B. ABA could improve the expression of GbEPSPs first, but deduce later. Different temperature treatments have different effects in the content of GbEPSP gene, and the highest expression at 42℃for 4h.(4) Molecular cloning, characterization and expression models of Cinnamoyl-CoA Reductase gene from G.biloba. Cinnamoyl-CoA Reductase (CCR, EC 1.2.1.44) catalyses the first step of the lignin pathway. The full-length cDNA sequences of CCR gene (designated as GbCCR) were isolated from G.biloba for the first time. The full-length cDNA of GbCCR is 1178 bp long and contains a 972 bp open reading frame (ORF) encoding a 323 amino acid protein. The deduced GbCCR protein showed high identities to other plant CCRs, and had closer relationship with Picea abies shares 56.3% homology. They both contain a common signature which is thought to be involved in the catalytic site of CCR. Phylogenetic tree analysis revealed that GbCCR shared the same ancestor with other CCRs, but the divergence time is early. Southern blot analysis indicated that GbCCR belonged to a multi-gene family. The expression analysis by RT-PCR showed that GbCCR had tissue specific manner in G.biloba, and the highest expression in stems and roots, the next in mature leaves, which were great difference with other flavones biosynthetic pathway gene. GbCCR was also found to be significant up-regulated by GA, but agrobacterium treatment had no significant change. The high level of GbCCR gene expression along the stalk suggests that the corresponding enzyme is probably involve in constitutive lignification.(5) Regulatory Element and Function Analysis of Chalcone Synthase Gene Promoter from Ginkgo biloba L. The regulative sequence (1711 bp) of chalcone synthase gene promoter (CHSP) from Ginkgo biloba L. was cloned by genomic walking. In bioinformatic analysis of sequence suggested that the sequence contained several typial cis-acting elements, including UV/blue light responsive elements, Phytohormone responsive elements, fungal elicitor responsive elements, MYB binding site, TATA-box and CAAT-box. A 1402 bp promoter sequence upstream 5'of translation start site of GbCHS were cloned and designated as GbCHSP, respectively. pBI121+CHSP and pBI121-35s were constructed and transformed into tobacco by LBA4404. These result showed that pBI121 and pBI121+CHSP both could drive the transient expression of GUS in tobacco and pBI121+CHSP expressed differentially in root, stem and leaf tissues of tobacco. Our discoveries will be help to understand the transcriptional regulatory mechanism on GbCHS expression and accumulation flavonoids.