Molecular breeding of transgenic solanaceous plants showing the resistance to Ralstonia solanacearum by the introduction of lysozyme gene

青枯病是青枯病的病原菌，主要危害青枯菌属植物。虽然人们已经采取了许多方法来控制这种病原菌，但还没有建立起一种可靠的保护植物的方法。在本研究中，为了培育对该病原菌具有遗传抗性的青枯病植株，我们计划从该细菌的噬菌体中分离出表达溶菌酶基因的转基因烟草植株。此外，我们还利用豌豆PSPAL1启动子来控制溶菌酶基因，该启动子具有根部特异性和病原菌诱导表达模式。从青枯雷尔氏菌噬菌体P4282中分离得到溶菌酶蛋白及相应基因，并对其进行了结构鉴定。我们构建了由PSPAL1启动子的调控元件(盒I、盒II和盒IV)组成的重复启动子，表达了极高的启动子活性。尤其是重复启动子在根中表现出比CaMV 35S启动子6.5的持活性。将CaMV 35S启动子、PSPAL1启动子或上述重复启动子分别与溶菌酶基因融合，转化烟草植株。整合了融合溶菌酶基因的转基因烟草植株表达了较高的裂解酶活性。通过接种35S：：Lux基因盒，将青枯病菌导入含有PSPAL1启动子：：GUS报告基因的转基因烟草植株，建立了同时观察寄主防御反应和病原菌行为的监测体系。

Ralstonia solanacearum is a causal agent of bacterial wilt mainly to solanacearum plants. Although many approaches were made to control this pathogen, a sure method to protect the plants was not established yet. In this study, to breed genetically resistant solanacearum plants to this pathogen, we planed to generate the transgenic tobacco plants expressing the lysozyme gene isolated from the bacteriophage of this bacterium. Furthermore we utilized pea PSPAL1 promoter which shows root spec and pathogen-inducible expression pattern for the control of lysozyme gene.We considered that the following results include the significant achievements in this research.1. From bacteriophage P4282 of Ralstonia solanacearum, we isolated lysozyme protein and correspond gene, and structurally characterized.2. We constructed the repeat promoter consisting the regulatory cis-elements (Box I, Box, II and Box IV of PSPAL1 promoter that expressed extremely high promoter activity. Especially the repeat promoter exhibited 6.5 hold activity than CaMV 35S promoter in roots.3. Each CaMV 35S promoter, PSPAL1 promoter or the repeat promoter described above was fused with lysozyme gene, and transformed tobacco plants.4. The transgenic tobacco plants that integrated chimeric lysozyme gene expressed higher lytic enzyme activity.5. By the inoculation of 35S :: Lux gene cassette introduced R. solanacearum on the transgenic tobacco plants possessing PSPAL1 promoter :: GUS reporter gene enabled to establish the monitoring system to investigate the host defense response and behavior of bacterial pathogen simultaneously.