Formation of antibacterial medicinal plants by transformation using lysozyme gene

将烟草（Nicotianatabacum）、曼陀罗（Daturainoxia）和野曼陀罗（Datanniaericosa）与根癌农杆菌（Agrobacteriumtumefaciens）菌株LBA 4404共培养，该菌株含有由溶菌酶和酵母转化酶信号肽构建的双元载体pBI 121，该双元载体携带可调控的CaMV 35 S启动子，能有效地将表达的溶菌酶分泌到胞间发挥抑菌活性。添加400 mg/l卡那霉素用于选择转化的愈伤组织和叶片段。(1)用溶菌酶基因引物对转溶菌酶基因和信号溶菌酶基因的烟草再生植株进行PCR检测，结果表明转溶菌酶基因的烟草再生植株分泌溶菌酶，而对照植株不分泌溶菌酶。(2)GUS表达最高的细胞系比未转化的愈伤组织的GUS活性高45.6倍。通过Southern杂交和组织化学分析确定转基因的性质。Western blot分析未检测到溶菌酶，但观察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之间溶菌酶表达存在差异。(3)体细胞胚、叶盘和茎尖作为感染段。除茎尖外，体细胞胚和叶盘系统均能获得再生植株。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.