摘要
通过菌落原位杂交和Southern杂交,从假单胞菌M18基因组文库中克隆了rpoS基因及相邻序列。为了深入研究影响rpoS基因表达的调控因素,运用同源重组技术,将无启动子β-半乳糖苷酶基因(-′lacZ)插入并融合于rpoS基因中,构建了假单胞菌M18rpoS基因突变株M18SZ。Miller法测定显示,突变株M18SZ的β-半乳糖苷酶可高达480U,而野生株检测不到β-半乳糖苷酶活性。表明,突变株中的rpoS基因与无启动子β-半乳糖苷酶基因已融合并且表达。在KMB培养基中生长量测定(OD600)的结果表明,突变株与野生株生长存在显著差异。
With hybridization in situ and Southern blots, an Eco RI- Xho I DNA fragment of 3. lkb in length containing an rpoS gene and its flanking sequence was first cloned into pBlueseript SK to generate pBLS by screening the genomic DNA library of Pseudomonas sp. M18. In order to identify the potential factors involved in rpoS gene expression and the regulatory mechanism truncated lacZ gene, of RpoS in strain M18, and a mutant named as the rpoS gene was inserted and fused in frame with a promoterless and M18SZ was then constructed through homologous recombination. Growth curves in KMB medium indicated that loss of RpoS made the mutant strain M18SZ more sensitive to alteration of some environmental factors. With detection and comparison of β-galactosidase activities from both the wild type strain M18 and its derivative M18SZ cultivated in KMB medium respectively, it was found that the expression level of β-galactosidase activities in the mutant M 18SZ was high and could come to 480U. Expression of β-galactosidase activities of the wild type strain M18 in KMB medium was not almost detected during its whole growth phase. With these results, it was confirmed that the rpoS gene did be fused in frame with the truncated lacZ gene in chromosome of the mutant M18SZ. Meanwhile, it is suggested that construction of a mutation, which is made with fusion in frame with the truncated lacZ gene, may be verified by detecting its β-galactosidase activity, not using Southern blot or PCR.
出处
《微生物学报》
CAS
CSCD
北大核心
2006年第5期709-713,共5页
Acta Microbiologica Sinica
基金
国家"十五"科技攻关项目(2001BA308A02-14)
国家自然科学基金项目(30370041)~~
