School of Life Sciences, Shandong University of Technology, Zibo, 255049, Shandong Province, China.
Key Laboratory of Coastal Biology and Biological Resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, 264003, China.
Mar Biotechnol (NY). 2020 Aug;22(4):572-580. doi: 10.1007/s10126-020-09978-z. Epub 2020 Jun 13.
The purpose of this study was to express an antimicrobial peptide in the chloroplast to further develop the plastid engineering of H. pluvialis. Homologous targeting of the 16S-trnI/trnA-23S region and four endogenous regulatory elements, including the psbA promoter, rbcL promoter, rbcL terminator, and psbA terminator in H. pluvialis, were performed to construct a chloroplast transformation vector for H. pluvialis. The expression of codon-optimized antimicrobial peptide piscidin-4 gene (ant1) and selection marker gene (bar, biolaphos resistance gene) in the chloroplast of H. pluvialis was controlled by the rbcL promoter and psbA promoter, respectively. Upon biolistic transformation and selection with phosphinothricin, integration and expression of ant1 in the chloroplast genome were detected using polymerase chain reaction (PCR), southern blotting, and western blotting. Using this method, we successfully expressed antimicrobial peptide piscidin-4 in H. pluvialis. Hence, our results showed H. pluvialis promises as a platform for expressing recombinant proteins for biotechnological applications, which will further contribute to promoting genetic engineering improvement of this strain.
本研究旨在将抗菌肽表达在叶绿体中，以进一步发展雨生红球藻的质体工程。通过同源靶向雨生红球藻的 16S-trnI/trnA-23S 区域和四个内源性调控元件，包括 psbA 启动子、rbcL 启动子、rbcL 终止子和 psbA 终止子，构建了一个用于雨生红球藻的叶绿体转化载体。经密码子优化的抗菌肽 piscidin-4 基因（ant1）和选择标记基因（bar，抗草丁膦基因）在叶绿体中的表达分别受 rbcL 启动子和 psbA 启动子的控制。通过弹道转化和草丁膦筛选，利用聚合酶链反应（PCR）、Southern 印迹和 Western 印迹检测 ant1 在叶绿体基因组中的整合和表达。采用该方法，我们成功地在雨生红球藻中表达了抗菌肽 piscidin-4。因此，我们的结果表明雨生红球藻有望成为表达重组蛋白的平台，用于生物技术应用，这将进一步有助于促进该菌株的遗传工程改良。
