白藜芦醇合酶基因的克隆及丹参的转化

目的为探索利用分子生物学技术培育新型药用植物的新途径,将外源的白藜芦醇合酶(RS)基因导入到丹参中表达,以改善或增加丹参的效用。方法根据已公布的核苷酸序列,利用引物悬挂延伸法,经过3次扩增,最终从葡萄基因组DNA中获得完整的RScDNA基因序列;以质粒pBin438为基础,构建含有RS目的基因的组成型植物表达载体。在根癌农杆菌介导下,利用叶盘法转化丹参,进而通过PCR、PCR-Southern杂交对不同的转基因植株进行筛选与检测。结果总共得到了45棵卡那霉素抗性植株,经PCR和PCR-Southern杂交检测,确定葡萄RS基因已整合到部分转基因丹参苗的基因组中。结论成功建立了白藜芦醇合酶基因转化丹参的体系,并获得了转基因植株。

Clone of resveratrol synthase gene and transformation of Salvia miltiorrhiza

Objective To explore a new method of producing novel medicinal plants by molecular biological technique.A resveratrol synthase(RS)gene was firstly expressed in Salvia miltiorrhiza for improvement and increase of the potential of S.miltiorrhiza.Methods Based on the published nucleotide sequences,the intact cDNA sequence of RS was obtained according to over-hang extension PCR protocol with grape genomic DNA as template through three times of amplifications.A plant component expression vector containing the RS gene was constructed,which was derived from plasmid pBin438.The RS gene was then introduced into S.miltiorrhiza with the leaf disc method mediated by Agrobacterium tumefaciens.The regenerated plants were assayed by PCR and PCR-Southern blotting analysis.Results The total 45 Kanamycin-resistant plants were obtained from this transformation.The results of PCR and PCR-Southern analysis confirmed that the RS gene had been integrated into the genome of some regenerated plants of S.miltiorrhiza.Conclusion An efficient transgenic system of S.miltiorrhiza by RS gene is established and some transgenic plants are obtained from this transformation.