Xie Xueqing, Tian Yuqi, Tian Jinghuan, Ning Wenyan, Wang Chunmei
School of Life Sciences, Beijing University of Chinese Medicine, Beijing 102488, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Nov 25;36(11):2467-2477. doi: 10.13345/j.cjb.200173.
The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.
外源基因在蓝藻中的低表达率是蓝藻基因工程的瓶颈之一。T7 RNA聚合酶表达系统已在大肠杆菌中实现了外源基因的高效表达。蓝藻和大肠杆菌都是革兰氏阴性菌，具有较高的遗传同源性。在蓝藻中构建T7 RNA聚合酶表达系统可能会提高外源基因的表达。为了在鱼腥藻PCC 7120中构建T7 RNA聚合酶表达系统，采用重叠延伸PCR和酶切连接技术等方法构建了位点特异性整合载体pEASY-T1-F1-TacT7RNAPCmR-F2和穿梭表达载体pRL-T7-hG-CSF。位点特异性整合载体能够表达T7 RNA聚合酶，穿梭表达载体在T7启动子驱动下表达hG-CSF。然后通过电穿孔将位点特异性整合载体导入野生型蓝藻，并通过三亲本接合转移将穿梭表达载体转入位点整合的转基因蓝藻中。最后，通过PCR鉴定蓝藻中外源基因的存在，通过RT-PCR检测蓝藻中外源基因的转录水平，并通过Western印迹检测蓝藻中外源基因的蛋白表达。成功构建了两种载体，T7 RNA聚合酶基因和hG-CSF基因顺利转入蓝藻，且两个基因均在蓝藻中表达。综上所述，在蓝藻中成功构建了T7 RNA聚合酶表达系统，hG-CSF基因的表达率比传统蓝藻表达系统提高了一倍。该表达系统将为蓝藻基因工程的应用提供更好的工具，并将推动蓝藻作为底盘细胞在未来合成生物学领域的发展。
