【摘要】 【目的】旨在构建一个用于粘球菌基因插入失活、同时可通过报告基因分析插入位点基因表达情况的质粒载体,并应用该质粒对黄色粘球菌MXAN1334基因功能和表达情况进行分析。【方法】通过PCR、酶切和连接等方法构建质粒载体pZCY11。从黄色粘球菌DK1622基因组上PCR扩增MXAN1334基因内部部分片段,插入载体pZCY11上lacZ基因的上游,构建重组质粒pZCY13,将其转入DK1622菌株,获得MXAN1334基因插入失活突变株ZC16-18。【结果】基因功能和表达情况分析质粒载体pZCY11含有抗性标记基因aph、自杀性质粒复制子OriR6K和无启动子的报告基因lacZ。突变株ZC16-18在CTT软硬琼脂平板上菌落扩展结果显示,MXAN1334基因插入失活会造成黄色粘球菌S运动能力缺陷。通过X-gal检测突变株ZC16-18中β-半乳糖苷酶酶活,实验结果显示,含有X-gal的平板上培养的ZC16-18菌落呈现蓝色,表明MXAN1334基因能够表达;颜色呈现的时间分析结果显示,MXAN1334基因表达时间较早。【结论】构建的质粒载体pZCY11不仅能够对目的基因进行功能的分析,而且能够同时通过报告基因分析基因的表达情况,可简化粘球菌中基因功能及表达情况的研究。更多还原

【Abstract】 [Objective] To construct a plasmid for analyzing gene functions and expressions and to study the MXAN1334 gene in Myxococcus xanthus with the plasmid. [Methods] We constructed the plasmid vector pZCY11,amplified MXAN1334 gene fragment from M. xanthus DK1622 by PCR,and inserted the fragment into a site upstream of lacZ,resulting in the recombinant plasmid pZCY13. The plasmid pZCY13 was transformed by electroporation to DK1622,producing a mutant ZC16-18 (ΔMXAN1334). [Results] The plasmid pZCY11 carried the resistance gene aph as the selectable marker,the replication origin of OriR6K and promoterless reporter gene lacZ. We examined the swarm expansions of ZC16-18 on CTT hard and soft agar,and the result indicated that MXAN1334 gene was probably involved in gliding motility in M. xanthus. In addition,β-galactosidase activity of ZC16-18 was detected by X-gal assay and the blue color developed was used to mark the colony growth. Time of colour showed that MXAN1334 gene was expressed in the early stage in M. xanthus. [Conclusion] The plasmid vector pZCY11 made it more convenient for the study on functions and the expressions of target gene in M. xanthus.更多还原