Formation of antibacterial medicinal plants by transformation using lysozyme gene

烟草(1)、曼陀罗(2)和地黄(3)与根癌农杆菌菌株LBA4404共培养，根癌农杆菌LBA4404含有用BOTN溶菌酶构建的双元载体pBI121和携带可调节的CaMV35S启动子的酵母转化酶信号肽，以有效地将表达的溶菌酶分泌到细胞间隙进行抗菌活性。(1)在烟草愈伤组织转化实验中，同时导入溶菌酶和信号溶菌酶基因的再生植株都能分泌溶菌酶，而对照植株不分泌溶菌酶。(2)GUS高表达株系的GUS活性是未转化植株的45.6倍。通过Southern印迹分析和组织化学方法鉴定转基因植株的性质。转化溶菌酶基因的愈伤组织和信号-溶菌酶嵌合基因转化的愈伤组织在蛋白印迹分析中未检测到溶菌酶的表达。(3)以体细胞胚胎、叶盘和茎尖为感染段。除茎尖外，体细胞胚和叶盘系统均可获得再生植株。然而，并不是所有的系统都能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.