质粒拯救型T-DNA标签质粒的构建

目的]为了高效率地获得转基因植物的旁邻序列。方法]以pUC18及pWM101为出发质粒,通过用限制性内切酶EcoRⅠ和HindⅢ双酶切质粒pUC18,得到含大肠杆菌复制起点及氨苄青霉素抗性基因的pUC18大片断,并将这段DNA整合到pWM101的T-DNA之中构建重组质粒。结果]该质粒利用根癌农杆菌转化植物后,可利用潮霉素筛选转化细胞,拯救质粒则可转化大肠杆菌后,以氨苄青霉素进行筛选。结论]该研究为以后的用T-DNA标签研究植物功能奠定了基础。

Construction of Rescue Plasmid with T-DNA Tag

Objective] The aim of the research was to obtain the flanking sequences in transgenic plants effectively.Method] With pUC18 and pWM101 as initial plasmids,double enzyme digestion was conducted on plasmid pUC18 with restriction endonucleases of EcoR Ⅰ and Hind Ⅲ to obtain large fragment of plasmid pUC18 that contained replication origin of E.coli and ampicillin resistance gene.The DNA fragment was integrated into T-DNA of plasmid pWM101 to construct a recombinant plasmid.Result] After transforming plant by using Agrobacterium tumefaciens,the plasmid could make use of hygromycin to screen the transformants.After transforming E.coli,the rescue plasmid could be screened by using ampicillin.Conclusion] The research laid the foundation for studying plant functions by using T-DNA tags.