对乳酸杆菌pPG质粒表达系统理想表达条件进行了探索。以干酪乳杆菌Lactobacillus casei 393为宿主菌,β-葡萄糖苷酸酶(gusA)基因作为表达的报告基因,根据干酪乳杆菌的生长特性对细胞表面表达载体pPG表达过程中各个关键因素的影响进行了...对乳酸杆菌pPG质粒表达系统理想表达条件进行了探索。以干酪乳杆菌Lactobacillus casei 393为宿主菌,β-葡萄糖苷酸酶(gusA)基因作为表达的报告基因,根据干酪乳杆菌的生长特性对细胞表面表达载体pPG表达过程中各个关键因素的影响进行了探索。得出最佳表达条件:将其培养于质量浓度为1%乳糖,初始pH值在6.0～6.5之间的MRS培养基中,30～32℃,静止培养至A590OD达0.5,培养时间约8 h,表达效率最高,外源蛋白的表达量可占菌体总蛋白的14%。展开更多
The constitutive promoter of cauliflower mosaic virus 35S （CaMV 35S） is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has...The constitutive promoter of cauliflower mosaic virus 35S （CaMV 35S） is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5＇ end of CaMV 35S promoter in the opposite orientation. To qualitatively and quantitatively estimate its bi-directional transcriptional function and activity, two visible reporter genes, gusA （13-glucuronidase, GUS） and gfp （green fluorescent protein, GFP）, were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences, respectively. Stable expression of gusA and gfp genes in transgenic tobacco （Nicotiana tabacum L.） was visulized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The expression of two reporter genes showed that the constructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations. The quantitative estimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excitation and emission. The GUS activity varied from g to 250 pmol 4-MU/min/mg protein and the GFP content varied from 0.9 to 1.8 μg/ mg protein in various lines of transgenic tobacco plants. Higher GUS activity generally coupled with lower GFP content, and vice versa.展开更多
文摘对乳酸杆菌pPG质粒表达系统理想表达条件进行了探索。以干酪乳杆菌Lactobacillus casei 393为宿主菌,β-葡萄糖苷酸酶(gusA)基因作为表达的报告基因,根据干酪乳杆菌的生长特性对细胞表面表达载体pPG表达过程中各个关键因素的影响进行了探索。得出最佳表达条件:将其培养于质量浓度为1%乳糖,初始pH值在6.0～6.5之间的MRS培养基中,30～32℃,静止培养至A590OD达0.5,培养时间约8 h,表达效率最高,外源蛋白的表达量可占菌体总蛋白的14%。
文摘The constitutive promoter of cauliflower mosaic virus 35S （CaMV 35S） is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5＇ end of CaMV 35S promoter in the opposite orientation. To qualitatively and quantitatively estimate its bi-directional transcriptional function and activity, two visible reporter genes, gusA （13-glucuronidase, GUS） and gfp （green fluorescent protein, GFP）, were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences, respectively. Stable expression of gusA and gfp genes in transgenic tobacco （Nicotiana tabacum L.） was visulized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The expression of two reporter genes showed that the constructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations. The quantitative estimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excitation and emission. The GUS activity varied from g to 250 pmol 4-MU/min/mg protein and the GFP content varied from 0.9 to 1.8 μg/ mg protein in various lines of transgenic tobacco plants. Higher GUS activity generally coupled with lower GFP content, and vice versa.
