Molecular cloning and characterization of GbMECT and GbMECP gene promoters from Ginkgo biloba.

Ginkgolides are key pharmaceutical components in Ginkgo biloba. Using the cDNA sequence of the MECP and MECT genes to design primers, we obtained the promoters of these genes from Ginkgo genomic DNA using the genome walking method. The two promoters were 744 and 982 bp in length, respectively. The cis-elements of the GbMECPs and GbMECT promoters were predicted and analyzed using the plant cis-acting regulatory element database. We found major cis-elements in the sequence of the GbMECT and GbMECPs promoters. The GbMECP promoter contains six TATA boxes and eight CAAT boxes. The GbMECT contains five TATA boxes and seven CAAT boxes. Furthermore, some cis-elements in the promoters of GbMECPs and GbMECT included hormone and light-regulated elements, UB-B-induced elements, and stress-related dehydration-responsive elements. Expression analysis results showed that the MECP gene is mainly involved in responses to CCC (cycocel) and UV-B, and that MECT is mainly involved in responses to wounding treatment. These results also showed that the expression model was consistent with the cis-elements present. During the annual growth cycle, the level of GbMECPs was significantly correlated with terpene lactones accumulation in leaves. A fitted quadratic curve showed the best model for correlating GbMECPs with terpene lactones in leaves. These results will help us to understand the transcriptional regulatory mechanisms involved in key gene expression and ginkgolide accumulation in G. biloba.

Functional characterization of the Ginkgo biloba chalcone synthase gene promoter in transgenic tobacco.

The regulative sequence (2273 bp) of the chalcone synthase gene promoter of biloba was cloned by genomic walking. A 2273-bp promoter 5' upstream translation start site of GbCHS was cloned and designated as GbCHSP. pBI121+CHSP:GUS and pBI121-35S:GUS were constructed and transformed into tobacco by LBA4404. We found that GbCHSP could drive transient expression of GUS in tobacco and differentially expressed in root, stem and leaf tissues of this plant. GUS activity regulated by the CHSP promoter were located in tissues (apical meristems) at the growing points of roots and stems. pBI121+CHSP:GUS could be induced by wounding, copper, UV-B, abscisic acid, and ethephon treatments of transgenic seedlings. This activity was weakly inhibited by gibberellin. Deletion analysis of the CHSP promoter in transgenic tobacco showed that CHSP1 complete promoter conferred a GUS expression and activity similar to that of 35 S(CaMV). GUS activity dropped dramatically when there were CHSP4, CHSP5 constructs and was almost totally absent when the CHSP6 construct was present. We conclude that the upstream sequence -1548 to -306 of GbCHSP is the main region for transcriptional regulation of the CHS gene and that it is activated by hormone and stress factors in G. biloba. These results will help us to understand the transcriptional regulatory mechanisms involved in GbCHS expression and flavonoid accumulation in G. biloba.

Molecular cloning and characterization of GbDXS and GbGGPPS gene promoters from Ginkgo biloba.

Ginkgolides are key pharmaceutical components in Ginkgo biloba leaves. 1-Deoxy-D-xylulose-5-phosphate synthase (GbDXS) and geranylgeranyl pyrophosphate (GbGGPPS) genes are critical genes involved in ginkgolide biosynthesis. In this study, the promoters of GbDXS and GGPPS, with 676 and 570 bp in length, respectively, were cloned by chromosome walking. The cis-elements of GbDXS and GbGGPPS promoters were predicted and analyzed by the plant cis-acting regulatory element (CARE) database. We found some major cis-elements in the sequence of GbDXS and GbGGPPS promoters. The GbDXS promoter has 3 TATA boxes, 10 CAAT boxes, 6 GATA boxes, and 1 I box. The GbGGPPS promoter has 1 TATA box, 6 CAAT boxes, 6 GATA boxes, and 4 I boxes. Furthermore, some stress-related cis-elements in the promoters of GbDXS and GbGGPPS were found to be light-regulated elements, including sequences over-represented in light-induced promoters (SORLIP1- AT), GATA box, and I box, a gibberellin-responsive element (WRKY), salicylic acid-induced (GT-1), cold- and dehydration-responsive (MYC-Core), and copper-inducible (CURE-Core). Further analyses of these cis-elements will aid in elucidating the molecular mechanisms regulating the expression of the GbDXS and GbGGPPS genes during ginkgolide accumulation in G. biloba.