Genetic Transformation Of The Key Genes In Ginkgolides Biosynthesis Pathway

Ginkgolides are pharmaceutical effective ingredients playing major part in the established medicinal functions of Ginkgo extracts,which have attracted great commercial interests as pharmaceuticals or nutraceuticals.However the contents of ginkgolides are very low in the native ginkgo plant materials and the ginkgo cell cultures hardly produce ginkgolides.Because of the expanding demands for ginkgo products and limited ginkgo tree resources,genetic modification of the ginkgolides biosynthesis pathway is becoming one of the most potential ways to increase ginkgolides content.Farnesyl diphosphate synthase(FPS) catalyzes two consecutive condensations of isopentenyl diphosphate with dimethylallyl diphosphate,and the resultant ger-anyl diphosphate.The ultimate product of these two reactions,arnesyl diphosphate(FPP), provides precursor for the biosynthesis of sterols,dolichols,sesquiterpene trilactone, prenylated proteins in plant.In Ginkgo biloba,it provides precursor for Bilobalide biosynthesis.1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(HDR) is proved to be the terminal-acting enzyme in the plastid MEP pathway which provides isoprenoid precursors for the biosynthesis of ginkgolides.FPS and HDR are the two key-enzymes in the ginkgolides biosynthesis pathway.So we choose the Gbfps and Gbhdr as the target genes for further study in metabolic engineering of ginkgolides.In order to investigate the effects of the two enzymes on the yields of ginkgolides in ginkgo calli,we construct two vectors of gene expression based on pCAMBIA1304 for transformation:Vector p1304+ Gbfps and Vector p1304+Gbhdr.These two genes were successfully transferred into embryos of Ginkgo biloba with Agrobacterium tumefaciens-mediated transformation method.After hygromycin(10 mg/L) selection, 32 and 8 hygromycin-resistant calli were obtained respectly.PCR analysis confirmed the Gbfps transgenic status of thirty independent callus lines and Gbhdr transgenic status of seven independent callus lines.Ginkgolides contents in 22 independent Gbfps transgenic callus lines and 4 independent Gbhdr transgenic callus lines were detected by HPLC-ELSD analysis respectly,and the result showed that ginkgolides content in line f-6 was significantly increased,reaching the 4.4 fold of the untransformed control line;ginkgolides content in line h-8 reached the 2.5 fold of the untransformed control line.