【摘要】 目的:构建含有大肠杆菌6-磷酸甘露糖异构酶(6-phosphomannose isomerase,PMI)基因并替换潮霉素抗性(hygromycin,hyg)基因的植物表达载体pCAMBIA1305-PMI,同时建立以PMI/甘露糖为筛选标记的白花丹参转基因体系。方法:首先从大肠杆菌Escherichia coli DH5α中克隆PMI基因,然后以PMI基因替换植物表达载体pCAMBIA1305中的hyg基因,构建了以PMI基因为选择标记基因的植物表达载体pCAMBIA1305-PMI,并用电击转化方法导入根癌农杆菌Agrobacterium tumefaciens LBA4404中,用叶片浸染法转化白花丹参。结果:在白花丹参MS+6-BA 1.5 mg·L-1+NAA 0.1 mg·L-1固体分化培养基中附加20 g·L-1甘露糖和10 g·L-1蔗糖为碳源的选择压力下,pCAMBIA1305-PMI的转化率为23.7%,对再生植株用PCR检测证实了PMI基因的导入。结论:建立了以PMI-甘露糖为选择系统的白花丹参转基因体系,可应用于后续目的基因的转化奠定了基础。更多还原

【Abstract】 Objective: To construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system. Method: The 6-phosphomannose isomerase gene( PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305,the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404,and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI. Result: Plant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g·L-1mannose and 10 g·L-1sucrose as a carbon source. The transformation efficiency rate was 23. 7%. Conclusion: Genetic transformation was confirmed by PCR,indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.更多还原