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天然药物合成生物学技术是一种采用多基因控制的方式,实现天然产物代谢途径在异源底盘细胞中的重构。启动子是调控基因表达的一种顺式元件,对多基因控制的代谢途径的平衡起着重要的作用。为了筛选获得性能优越的启动子用于多基因控制的代谢途径,一个基于红色荧光蛋白的酵母检测质粒被构建。首先,根据已经发表的红色荧光蛋白基因,设计并合成了一系列引物,通过连续重叠PCR的方法合成了全长红色荧光蛋白基因DsRed-Monomer,并将该红色荧光蛋白基因导入酿酒酵母,SDS-PAGE、Western blot以及荧光显微镜观察都表明该红色荧光蛋白基因在酿酒酵母中获得了表达。然后,将具备功能的DsRed-Monomer基因与酿酒酵母表达载体pGBT9进行重组,获得能进行启动子文库筛选的启动子检测质粒pGBT9Red。为了检测该质粒的效能,通过PCR从酿酒酵母基因组中克隆得到了6种启动子(包括4种诱导型启动子和2种组成型启动子),并将6种启动子分别克隆到pGBT9Red中,置于红色荧光蛋白基因DsRed-Monomer上游,通过荧光成像确定启动子对红色荧光蛋白基因表达的调控效率。结果表明,6种启动子(GAL1、GAL2、GAL7、GAL10、TEF2和PGK1)在酿酒酵母中均能调控DsRed-Monomer的表达。该检测质粒的成功构建为进一步进行启动子活性分析和启动子元件文库筛选奠定基础。
Natural medicine synthesis biology technology is a multi-gene control approach to achieve the natural product metabolic pathways in allogenic cells of the reconstitution. Promoters are cis-elements that regulate gene expression and play an important role in the balance of metabolic pathways controlled by multiple genes. To screen for a superior promoter for the multigene-controlled metabolic pathway, a red fluorescent protein-based yeast detection plasmid was constructed. First of all, a series of primers were designed and synthesized according to published red fluorescent protein genes. The full-length red fluorescent protein gene DsRed-Monomer was synthesized by continuous overlapping PCR. The red fluorescent protein gene was introduced into Saccharomyces cerevisiae, SDS- PAGE, Western blot and fluorescence microscopy showed that the red fluorescent protein gene was expressed in Saccharomyces cerevisiae. Then, the functional DsRed-Monomer gene was recombined with the Saccharomyces cerevisiae expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red capable of screening the promoter library. To test the efficiency of the plasmid, six promoters (including four inducible promoters and two constitutive promoters) were cloned from Saccharomyces cerevisiae genome by PCR and six promoters were cloned into pGBT9 Red, Placed upstream of the red fluorescent protein gene DsRed-Monomer, and the regulatory efficiency of the promoter on the red fluorescent protein gene expression was confirmed by fluorescence imaging. The results showed that all six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) could regulate the expression of DsRed-Monomer in Saccharomyces cerevisiae. The successful construction of the test plasmid laid the foundation for further analysis of promoter activity and screening of the promoter element library.