The purpose of this study was to establish a plastid transformation system for expressing recombinant proteins in Nannochloropsis gaditana. On the basis of the sequenced plastid genome, the homologous flanking region, 16S‐trnI/trnA‐23S, and the endogenous regulatory fragments containing the psbA promoter, rbcL promoter, rbcL terminator, and psbA terminator were amplified from N. gaditana as elements of a plastid transformation vector. Then, the herbicide‐resistant gene (bar) was used as a selectable marker, regulated by the psbA promoter and rbcL terminator. Finally, two codon‐optimized antimicrobial peptide‐coding genes linked by endogenous ribosome binding site (RBS) in a polycistron were inserted into the constructed vector under the regulation of the rbcL promoter and psbA terminator. After microparticle bombardment, the positive clones were detected using polymerase chain reaction (PCR), and Southern and Western blotting were used to assess the co‐expression of the two antimicrobial peptides from the plastid. Nannochloropsis gaditana showed the potential to express recombinant proteins for biotechnological applications, for example, for the development of oral vaccines in aquaculture.

中文翻译：

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海洋藻类Nannochloropsis gaditana的质粒工程，用于两种重组肽的共表达

本研究的目的是建立一个表达拟南芥（Nannochloropsis gaditana）重组蛋白的质体转化系统。根据测序的质体基因组，从N扩增出同源侧翼区域16StrnI / trnA- 23S以及包含psb A启动子，rbc L启动子，rbc L终止子和psb A终止子的内源性调控片段。 gaditana作为质体转化载体的元素。然后，将抗除草剂基因（bar）作为选择标记，由psb A启动子和rbc调控L终结者。最后，在rbc L启动子和psb A终止子的调控下，将通过多顺反子中的内源核糖体结合位点（RBS）连接的两个密码子优化的抗菌肽编码基因插入到构建的载体中。微粒轰击后，使用聚合酶链反应（PCR）检测阳性克隆，并使用Southern和Western印迹法评估来自质体的两种抗菌肽的共表达。Nannochloropsis gaditana表现出表达重组蛋白的潜力，可用于生物技术应用，例如用于开发水产养殖中的口服疫苗。