Formation of antibacterial medicinal plants by transformation using lysozyme gene

将烟草(1)、牛油果(2)和地黄(3)与农杆菌（Agrobacterium tumefaciens）菌株LBA4404共培养，该菌株含有由植物溶菌酶和酵母转化酶信号肽构建的双载体pbi121，该载体携带可调节的CaMV35S启动子，可将表达的溶菌酶有效分泌到细胞间隙，发挥抑菌活性。卡那霉素添加量为400 mg/l，用于愈伤组织和叶段的选择。(1)在烟草实验中，同时携带溶菌酶和信号溶菌酶基因的再生植株均分泌溶菌酶，利用溶菌酶基因引物进行PCR检测，而对照植株不分泌溶菌酶。(2) GUS最高表达系的GUS活性是未转化愈伤组织的45.6倍。通过southern blot和组织化学分析确定转基因性质。western blot分析未检测到溶菌酶，但检测到溶菌酶基因转化的愈伤组织与信号-溶菌酶嵌合基因转化的愈伤组织溶菌酶的表达有差异。(3)以体胚、叶片和茎尖为感染段。除茎尖外，再生植株体胚和叶片系统均可再生。然而，并不是在所有的系统中都检测到溶菌酶的分泌。

Nicotiana tabacum (1), Datura inoxia (2) and Rehmannia glutinosa (3) have heen co-cultivated with Agrobacterium tumefaciens strain LBA4404 which contained binary vector pBI121constructed using botn lysozyme and yeast invertase signal peptide carrying a regulatable CaMV35S promoter for effective secretion of expressed lysozyme to the intercellular spaces for antibacterial activity. The addition of 400 mg/l kanamycin was used for the selection of transformed callus and leaf segments.(1) In the experiments of Nicotiana tabacum, the regenerated plantlets transformed with both lysozyme and signal-lysozyme genes secreted lysozyme which was determined by PCR using primer of lysozyme gene although the control plantlets did not secrete lysozyme.(2) The highest line of GUS expression showed 45.6 times of GUS activity compared to untransformed callus. The transgenic nature was determined by the southern blot analysis and the histochemical assay. Lysozyme was not detected by the western blot analysis, although differences of lysozyme expression between the callus transformed with lysozyme gene and that transformed with signal-lysozyme chimeric gene was observed.(3) Somatic embryos, leaf disks and shoot tips were used as infection segments. Regenerated plantlets were obtained both somatic embryo and leaf disk systems except that of shoot tip. However, the secretion of lysozyme has not been detected in all systems.