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.

中文翻译：

---

表达抗微生物肽的单细胞绿藻雨生红球藻的叶绿体基因工程。

本研究的目的是在叶绿体中表达一种抗菌肽，以进一步开发雨生红球藻的质体工程。对雨生红球藻中的16S- trnI / trnA -23S区和4个内源调控元件进行同源靶向，包括psbA启动子、rbcL启动子、rbcL终止子和psbA终止子，构建雨生红球藻叶绿体转化载体。密码子优化的抗菌肽piscidin -4 基因 ( ant1 ) 和选择标记基因 ( barH. pluvialis叶绿体中的 biolaphos 抗性基因）分别由rbcL启动子和psbA启动子控制。在用膦丝菌素进行基因枪转化和选择后，使用聚合酶链反应 (PCR)、Southern 印迹和蛋白质印迹检测叶绿体基因组中ant1 的整合和表达。使用这种方法，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用于生物技术应用的重组蛋白的平台，这将进一步促进该菌株的基因工程改良。