【摘要】 丁香假单胞杆菌1336致病菌缺失pcs基因后不能合成磷脂酰胆碱(PC),也不能诱发植物的超敏反应(HR).从它的基因组中分别克隆出25个T3SS-相关基因,包括编码组件蛋白和表达调控基因.在E.coli中成功表达了其中19个基因,并且纯化出16种蛋白.在野生型和pcs~-突变型中,无论是编码T3SS组件蛋白基因、调节基因还是效应蛋白基因的转录水平都没有呈现显著性差异.与野生型比较,1336 pcs~-突变型细胞质中14个T3SS-关联蛋白的相对量也没有显著性差异,但细胞膜中的HrpB、HrpE、HrpF和Hrc J相对量都减少约10%.36%T3SS组件蛋白同时减少可能导致T3SS装置不完整,而这种有缺陷的T3SS装置不能行使正常生理功能.由此可见,膜磷脂中的PC在丁香假单胞杆菌T3SS装置组装过程中起着十分重要的作用.更多还原

【Abstract】 Pseudomonas syringae pv. syringae van Hall CFCC 1336( pss1336) was unable to elicit hypersensitive response( HR) after its phosphatidylcholine synthase gene( pcs) was deleted. Twenty-five genes,including regulatory genes and those genes encoding protein components of T3 SS,were cloned from the genomic DNA of pss1336. Nineteen genes were expressed in E. coli,in which 16 proteins were successfully purified to homogeneous. Analysis by semi-quantitative RT-PCR showed that the transcriptional level of 34 genes including regulatory genes,protein-component genes and effector genes did not exhibit any significant difference( p > 0. 05) between the 1336 wild type and the 1336 pcs~-mutant. Analysis by western blotting showed that relative quantities of 14 T3 SS-associated proteins were similar to those for the 1336 wild type. In contrast,the relative quantity for HrpB、HrpE、HrpF and Hrc J in the membrane of 1336 pcs~-mutant decreased approximately 10% as compared to the 1336 wild type. Thirty-six percent protein components simultaneously decreased more than 10% in bacterial membranes,which resulted in forming an imperfect T3 SS apparatus.Clearly,PC plays an important role in the assembly of T3 SS apparatus in pss1336.更多还原