银杏GGPPS转运肽与GFP融合基因表达载体的构建

[目的]构建银杏GGPPS转运肽与GFP融合基因表达载体。[方法]以银杏为材料,采用DNA重组技术克隆GGPPS基因质体转运肽（TP）序列,并将其与高效植物表达载体p1304＋连接形成融合表达载体（p1304＋-TP）;冻融法转化根瘤农杆菌EHA105,构建工程菌（EHA105-p1304＋-TP）。[结果]成功构建了银杏GGPPS转运肽与GFP融合基因表达载体及农杆菌工程菌。[结论]为进一步研究TP转运肽的亚细胞定位奠定基础,有助于阐明银杏内酯前体生物合成关键步骤的分子机理,同时为银杏内酯的代谢工程研究提供重要依据。

The Construction of the Fuse Gene Expression Vector which Consisted of GFP and TP Gene of GGPPS from the Ginkgo biloba L.

[Objective] The aim was to construct the fuse gene expression vector which consisted of GFP and TP gene of GGPPS from the Ginkgo biloba L. [Method] The transit-peptide (TP) sequence of GGPPS from Ginkgo biloba L. cDNA was successfully cloned by using DNA recombination technology,which was then connected to efficient plant expression vector P130+ to construct the the fuse gene expression vector p1304+-TP. Then engineering strain EHA105-p1304+-TP was constructed by transforming p1304+-TP to Agrobacterium rhizogenes EHA105 using freeze-thaw method. [Result] The fuse gene expression vector which consisted of GFP and TP gene of GGPPS from the Ginkgo biloba L. and engineering strain EHA105-p1304+-TP were successfully constructed. [Conclusion]It laid a foundation for further study on subcellular localization of TP transit peptide,and will also help to clarify the the molecular mechanism of key step in biosynthesis of ginkgolides precursors,meanwhile,it will provide an important basis for research on metabolic engineering of ginkgolide.