粘球菌基因功能和表达情况分析质粒pZCY11的构建及其应用

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

摘要:

摘要：【目的】旨在构建一个用于基因插入失活，并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DK622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。

Abstract:

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 (?MXAN1334). [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.