粘球菌基因功能和表达情况分析质粒pZCY11的构建及其应用

【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。

Construction and application of plasmid pZCY11 for analyzing gene functions and expressions in Myxococcus

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 (AMXAN1334). 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.