Clonging Of MECT, MECPs Gene Promoters From Ginkgo Biloba L. And Construction Of Plant Expression Vector

Ginkgo terpene lactones is an important traditional medicinal ingredient that extractedfrom ginkgo biloba leaves, The research shows that it can protect the central nervoussystem, reduce ischemic injury, antishock, antiallergic and so on. Now the studies onincreasing the content of the terpene lactone in G.biloba have became more and morepopular. In order to know more about the physiological and biochemical metabolism ofthe terpene lactone and to increase the content of the terpene lactone via geneticengineering in the near future, we obtained two key genes GbMECT, GbMECPs promoterfrom tall fescue genomic DNA by genome walking which involved in the accumulativeprocess of the terpene lactone. After the sequence analysis of these two promoters, we alsoconstructed a plant expression vector pBI121+GbMECPs to lay a foundation of functionanalysis of this promoter. The main results are as follows:(1) Molecular cloning and Regulatory Element Analysis of2-C-methyl-D-erythritol4-phosphate Cytidyltransferase Gene Promoter from Ginkgo bioba L. The regulativesequence (982bp) of GbMECT was cloned by genome walking. In bioinformatic analysisof sequence suggested that the sequence contained several types of cis-acting elements,including typical light responsive elements, such as GATA-box (GATA), GT-1motif(GRWAAW) and I-box (GATAA); cytokinins responesive elements: ARR1(NGATT) andCPBCSPOR (TATTAG), ABA responesive elements: MYC (CANNTG) and MYB1AT(WAACCA), gibberellin responesive elements: WRKY-motif (TGAC), GARE (TAAC-AGA) and GA-element (TAACAAR), auxin responesive element: ASF-1motif (TGACG)and ethephon responesive element: ACS (TAAAATAT), theses elements arePhytohormone responesive elements. The stress resistance elements are as follows: thedisease resistance element TGTCA sequence, the wounding responesive element ERF3(TGACY)and the Heat shock element HSE-motif (CCAAT), There are some other typeslike copper-response elements, amylase elemnts and so on. Theses characteristics fullyshows the efficiency and the complexity of gene promoter regulation at the transcriptionlevel.(2) Molecular cloning, Regulatory Element Analysis and Plant Expression Vector Constructe of2-C-methylerythritol2,4-cyclodiphosphate synthase Gene Promoter fromGinkgo bioba L. The regulative sequence (744bp) of GbMECPs was cloned by genomewalking. In bioinformatic analysis of sequence suggested that the sequence containedseveral TATA and CAAT boxes, as well as some typical light-regulated elements, such asGT-1motif (GRWAAW), Circadian box (CAANNNNATC) et al. Furthermore, there arevarious kinds of hormone-regulated elements that response to auxin, gibberellin, ethephon,cytokinins and abscisic acid, and some elements related to plant stress responses thatmediated by MYB, MYC transcription factor. By replacing the35S promoter, GbMECPspromoter was inserted upstream of the gus reporter gene in plasmid pBI121, and the plantexpression vector pBI121+GbMECPs was constructed. Agrobacterium tumefacientsmediated method will be used to transform the recombinant plasmid into tobacco, in orderto lay a foundation for further research about tissue-specific expression and functionanalysis of GbMECPs promoter.