外源基因的表达效率低是蓝藻基因工程发展的瓶颈之一，T7 RNA聚合酶表达系统实现了大肠杆菌中外源基因的高效表达，蓝藻与大肠杆菌同为革兰氏阴性菌，具有较高的遗传同源性，在蓝藻中构建T7 RNA聚合酶表达系统有可能提高外源基因在蓝藻中的表达效率。为了在鱼腥藻7120中构建T7 RNA聚合酶表达系统，采用重叠延伸PCR技术和酶切连接等方法构建能够表达T7 RNA聚合酶的定点整合载体pEASY-T1-F1-TacT7RNAPCmR-F2以及由T7启动子驱动hG-CSF基因表达的穿梭表达载体pRL-T7-hG-CSF；采用电击转化法将定点整合载体导入野生型鱼腥藻中，通过三亲接合的方法将穿梭表达载体转入已定点整合T7 RNA聚合酶的转基因鱼腥藻中。利用PCR技术鉴定外源基因在蓝藻中的存在；RT-PCR方法检测外源基因在蓝藻中的转录情况；Western blotting实验检测外源基因在蓝藻中的蛋白表达情况。结果表明两种载体构建成功，T7 RNA聚合酶基因和hG-CSF基因被转入鱼腥藻中，两个基因均在藻中表达，T7 RNA聚合酶表达系统在鱼腥藻中构建成功，与传统蓝藻表达系统相比，文中在鱼腥藻中构建的T7表达系统使hG-CSF基因的表达量提高2倍。该表达系统将为蓝藻基因工程的应用提供更优的工具，将促进蓝藻作为底盘细胞在合成生物学等领域的发展。
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.
北京中医药大学发展基金 (No. 2020071420021)，国家自然科学基金 (No. 81774014) 资助。