农杆菌介导银杏遗传转化体系的建立及Gb-DXR基因表达载体的构建

Establishment of the Agrobacterium-mediated Genetic Transformation System of Ginkgo biloba L. and the Construction of the Expression Vector of Gb-DXR

[目的]为提高农杆菌介导的银杏遗传转化效率提供参考。[方法]以成熟的银杏种子胚为外植体,在无激素MS培养基上预培养48h后,采用3种农杆菌介导将GUS基因导入银杏胚中,经过组织化学染色法检测到GUS瞬时表达活性,对影响GUS基因表达的因素进行初步分析;并构建了银杏内酯前体合成途径上1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR)的表达载体。[结果]该研究得到较合适的遗传转化方案,即用银杏胚作为外植体,用携带pCAMBIA1304+的EHA105农杆菌进行侵染,共培养3d,进行GUS染色,结果显示转化后GUS阳性率较高。[结论]该研究为进一步的银杏转基因研究工作奠定了基础。

[Objective]The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba L.mediated by Agrobacterium.[Method]Taking the mature embryos of Ginkgo biloba L.seeds as explants,after 48 hours’ pre-cultivation on MS medium in the absence of hormone,GUS gene was transmitted into embryos of Ginkgo biloba L.mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the synthesis approach of lactone precursor of Ginkgo biloba L.was constructed.[Result]A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba L.as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,culture for 3 days and GUS staining.The results showed that GUS positive rate after transformation was higher.[Conclusion]The research laid the foundation for further study on the transgene of Ginkgo biloba L..