Yao Zhen-Sheng, Chen Zhong-Yi, Chen Zhi-Yi, Zheng Xiao-Bo, Zhang Jie, Huang Da-Fang
State Key Lab for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection Chinese Academy of Agricultural Science, Beijing 100094, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Sep;19(5):551-5.
The full length sequence of the promoter and gfp gene were obtained respectively by PCR with two pairs unique primers PxyF/R and primers gfpF/R, which were designed according to the gfp gene and promoter sequence of xylase operon from Bacillus subtilis 168, and the DNA template plasmids pHT315-xyIR and pGFPuv. Furthermore, the fused translational expression cassette PxylR-gfp was constructed using overlapping PCR technique with the primers pair PxyF/gfpR and the mixture of above PCR production. After being digested by Kpn I and Sph I , PxylR-gfp expression cassette was inserted into E. coli-B. thuringiensis shuttle vecter pHT315 and E. coli-B. subtilis shuttle vecter pRP22, and the resulted recombinant plasmids were named as pGFP315 and pGFP22 respectively. Both recombinant plasmids were transferred into B. subtilis lab strain 168 and the resulted transformants are bright green performance under 365 nm UV light. However, only pGFP22 can be introduced into the natural strain B916. The transformants containing pGFP22 have bright green performance under 365 nm UV light and was named B916-gfp. Antifungal activities testing results proved that there is no obvious difference between B916 and the engineered strains B916-gfp. Research results also showed that the stability of B916-gfp was 94% after growth about 175 generations at 37 degrees C, and the losing rate of plasmid was less than 3.5 x 10(-4) per generation.
分别使用两对独特的引物PxyF/R和引物gfpF/R通过PCR获得启动子和gfp基因的全长序列，这两对引物是根据枯草芽孢杆菌168木聚糖酶操纵子的gfp基因和启动子序列以及DNA模板质粒pHT315 - xyIR和pGFPuv设计的。此外，使用引物对PxyF/gfpR和上述PCR产物混合物通过重叠PCR技术构建融合翻译表达盒PxylR - gfp。经Kpn I和Sph I酶切后，将PxylR - gfp表达盒插入大肠杆菌 - 苏云金芽孢杆菌穿梭载体pHT315和大肠杆菌 - 枯草芽孢杆菌穿梭载体pRP22，得到的重组质粒分别命名为pGFP315和pGFP22。将这两种重组质粒转入枯草芽孢杆菌实验室菌株168，所得转化子在365 nm紫外光下呈现亮绿色。然而，只有pGFP22能够导入天然菌株B916。含有pGFP22的转化子在365 nm紫外光下呈现亮绿色，命名为B916 - gfp。抗真菌活性测试结果证明B916与工程菌株B916 - gfp之间没有明显差异。研究结果还表明，B916 - gfp在37℃下生长约175代后的稳定性为94%，质粒丢失率每代小于3.5×10(-4)。
