PMI基因作为选择标记的植物表达载体构建及转化白花丹参体系的建立

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。

Construction of plant expression vectors with PMI gene as selection marker and their utilization in transformation of Salvia miltiorrhiza f.alba

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^-1 mannose and 10 g·L^-1 sucrose 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.