银杏内酯合成二萜环化酶基因克隆与生物转化研究

Ginkgo biloba trees contain specific diterpene compounds, gikgolkides which are present in tissues as trace amounts (1-3μg g-1 FW). Some of the beneficial pharmacological effects have been attributed to the gikgolides that are highly specific platelet-activating factor antagonists. To develop a method for producing the structurally unique family of diterpenoids, the cDNA coloning of ditepene cyclase from Ginkgo biloba has been considered as a first step. The homology-based PCR coloning strategy was used to clone the gene. 3 cDNA libraries were constructed using leaves, bark and roots. 24 primers were used to run PCR with different combinations to have conservative or 5',3' fragments. Chromosomal walking method also used to have more sequence informations . From cDNA libraries and genomic DNA, 82bp conservative and 417bp fragments were obtained and sequenced. The sequences showed 98% similarity with the one from Taxus chinenses. During the project time, a diterpene cyclase from Ginkgo biloba was published. According to the paper and our results, many experiments have been carried out to clone the gene including PCR, cDNA librarie screening and chromosomal walking. Unfortunately, no longer cDNA fragments obtained from Ginkgo biloba based on the paper information and neither our preliminary work. For the better results, RACE methods and more fragments sequencing will be performed continuously. The progress report will be sent to NSFC in future.

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