Molecular Cloning And Transformation Of Genes Involved In The Biosynthetic Pathways Of Flavonoids Of Ginkgo Biloba L.

Ginkgo boliba L.is ancient and precious in plant kingdom.It's usually called the "living fossil".Because of containing the active ingredients like flavonoids and terpenoids,ginkgo is thought to be valuable as medicine.In order to deepen the research in the biosynthetic pathways of flavonoids and to lay a groundwork for increasing contents of ginkgo flavonoids by using bioengineering in the near future, this text presents for the first time the cloning and characterization of two key genes, GbC4H coding for cinnamate 4-hydroxylase and GbC3H coding for p-coumarate 3-hydroxylase.Cinnamate 4-hydroxylase is involved in the general phenylpropanoid pathway upstream of the biosynthetic pathway of flavonoids,while p-coumarate 3-hydroxylase is involved in the lignin biosynthesis pathway,a pathway collateral to the one for the biosynthesis of flavonoids.GbC4H,GbC3H and GbCHS,GbFLS (coding for chalcone synthase and flavonol synthase respectively,also cloned by our lab)were transformed into tobacco and the callus of ginkgo to confirm the functions of the genes and the influences to the content of flavonoids.To study the influence of the regulatory factors on the biosynthesis of flavonoids,a WRKY gene,which may be involved in the biosynthesis of flavonoids,was also cloned from Brassica chinensis (BcWRKY)and transformed into tobacco alone or together with the genes mentioned above.Cinnamate 4-hydroxylase(C4H)catalyzes the second committed step in the general phenylpropanoid pathway.The full-length cDNA of C4H gene from G.biloba (designated as GbC4H)and its genomic DNA sequence were obtained by using RACE and PCR.The comparison between cDNA and genomic DNA sequences revealed that GbC4H was composed of two exons and one intron.GbC4H was found to have extensive homology with other plant C4H proteins via multiple alignments. Phylogenetic tree analysis revealed that GbC4H had closer relationship with C4Hs from gymnosperm plant species than from other plant species.Southern blot analysis indicated that GbC4H belonged to a multigene family.RT-PCR analyses revealed that GbC4H expressed differentially in the root,stem and leaf and the expression could be induced by salt,mannitol,cold and drought treatments.The plant expression vector p1304-GbC4H containing GbC4H was also constructed for plant transformation.P-coumarate 3-hydroxylase(C3H)is a key enzyme for the biosynthesis of lignins.The full-length cDNA and genomic DNA sequences of C3H gene were isolated from G.biloba(designated as GbC3H)using RACE and PCR.The comparison between cDNA and genomic DNA sequences revealed that GbC3H was composed of three exons and two introns.The deduced GbC3H protein showed high identities to other plant C3Hs via multiple alignments.Phylogenetic tree analysis revealed that GbC3H had closer relationship with C3Hs from gymnosperm plant species than from other plant species.Southern blot analysis indicated that GbC3H belonged to a multi-gene family.RT-PCR analysis revealed that GbC3H gene expressed in root and leaf,but not in the stem.The plant expression vector PBI121-GbC3H containing GbC3H was also constructed for plant transformation.The WRKY proteins are a superfamily of transcriptional regulators that appear to be unique to plants.The full-length cDNA and genomic DNA sequences of BcWRKY were isolated from Brassica chinensis using RACE and PCR.The comparison between cDNA and genomic DNA sequences revealed that BcWRKY was composed of five exons and four introns,the same with the genes of other regulators.The 5' flanking region of BcWRKY was isolated by genome walking technique,and the W-box which could be recognized by WRKY proteins was found in this region.Some main cis-acting elements including TATA box and stress-responsiveness elements were predicted and analyzed.Multiple alignment analysis showed that the WRKY domain of BcWRKY had high identities to those from other plant species. Homology-based structure modeling showed that the WRKY domain of BcWRKY had a same 3D model with that of AtWRKY4.RT-PCR analysis showed that BcWRKY expressed in root,stem and leaf at different levels and the expression of BcWRKY were induced by wounding,SA and bacteria treatments.The BcWRKY protein was successfully expressed in E.coli with the molecular weight predicted by bioinformatics.Western blot analysis indicated that the rBcWRKY containing His-tag had specific immune activity with anti-His antibodies.The plant expression vector p35S-2300-BcWRKY containing BcWRKY was also constructed for plant transformation.The above-mentioned genes and their characterization will provide a solid base for further studies on the biosynthetic pathways of flavonoids in G biloba,at molecular and biochemical levels.The genes also make it possible to bioengineering ginkgo flavonoids for human health in the future.