假单胞菌表达载体pYMB03的构建与性能分析  

Construction and Characterization of Pseudomonas Shuttle Expression Vector pYMB03

作　　者：叶婷[1] 姚陈[1] 李茜茜[1] 张红星[1] 李林[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070

出　　处：《应用与环境生物学报》2008年第4期528-533,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(No. 30370026)资助~~

摘　　要：恶臭假单胞菌(Pseudomonas putida)是具有强抗逆性能的环境优势菌,构建其质粒表达载体有着明显的应用潜力.将恶臭假单胞菌AB92019菌株中肽聚糖相关脂蛋白编码基因的启动子PoprL和质粒载体pTrcHis-B的多克隆位点片段插入到质粒载体pUCP18的EcoRI/HindIII位点,获得了重组载体pYMB03.用绿色荧光基因gfp作为标记进行外源蛋白表达的结果表明,该载体能分别在恶臭假单胞菌AB92019菌株和大肠杆菌DH5a菌株中,由启动子PoprL启动组成型表达GFP蛋白并使细胞产生可见荧光.经SDS-PAGE验证,所产生的GFP蛋白分别占细胞总蛋白的12.5%和5.0%.重组菌株YMB001中GFP表达量与菌体培养时间有关,在稳定期后期其相对荧光强度达到最大值(D600nm=1.0),但与培养温度未见相关性.对携带该载体的2株重组恶臭假单胞菌7次168h继代培养测定,载体pYMB03的稳定性均为100%.Pseudomonas putida is a kind of dominant bacteria known to survive in the contaminated environments. To develop a plasmid-based expression vector to facilitate the genetic manipulation, in the present study, a 380-bp of PCR-amplified promoter sequence of peptidoglycan-associated fipoprotein encoding gene （PoprL） from P. putida AB92019, together with a MCS fragment from Escherichia coli plasmid vector pTrcHis-B, was inserted into EcoRI/HindIII sites of plasmid vector pUCP18 to generate a Pseudomonas-Escherichia shuttle expression vector pYMB03. The expression efficiency of pYMB03 was further investigated using the green fluorescence protein （GFP） as the reporter, which allowed facilely determination of the specific fluorescence intensity in the recombinant strains. It showed that GFP could be constitutively expressed either in the recombinant P. putida YMB001 or in the transformed E. coli DHSa strain that harbored the recombinant plasmid pYMBP, which was initiated by the introduced promoter （PoprL） of pYMB03. As a result, the host strains were illuminant that was observable through the optical fluorescent microscopy. The SDS-PAGE profile indicated that the expressed GFPs comprised of approximately 12.5% and 5% of the total intracellular proteins in YMB001 and the transformed E. coli DHSα, respectively. It also showed that the GFP expression level in YMB001 was affected by culture time rather than by culture temperature, and a maximum GFP fluorescent intensity could be detectable when cells were growing at the stationary phase. Additionally, by directly enumerating the grown colonies in the antibiotic-free plates, it was showed that the stability of the shuttle expression vector pYMB03 was 100% when inoculated in succession for 7 times and cultured for 168 h. Fig 7 , Tab 1, Ref 17

关 键 词：表达载体 假单胞菌 启动子 构建 性能 

分 类 号：Q782[生物学—分子生物学]