The eukaryotic green microalga Tetraselmis suecica is commonly used for aquaculture purposes because of its high stress tolerance and ease of culture in a wide spectrum of environments; they are therefore suitable candidates for biotechnology applications. To date, no data are available regarding chloroplast transformation vectors based on specific endogenous promoters and homologous targeting regions. We report on the identification of Tetraselmis suecica genes encoding the ribulose bisphosphate carboxylase/oxygenase large subunit protein, the photosystem II D1 protein and the ATP synthase CF1‐beta subunit protein together with their untranslated regions (5′UTR, 3′UTR). The full‐length ORFs of the putative genes with their regulatory sequences were obtained. We were also able to identify the downstream 3′ end of the large subunit ribosomal RNA gene (23S) along with the 5S RNA end‐to‐end with the psbA gene on the complementary strand. The intergenic region between these genes appears to be a good target site for the integration of target proteins. Moreover, we identified a back‐to‐back promoter region among the rbcL and atpB genes. To assess the bidirectionality activities of both promoters, a dual reporter vector was constructed for Tetraselmis suecica transformation containing the cat and TurboGFP genes driven by the 5′rbcL/5′atpB divergent promoter. The vector included the 23S‐5S and psbA nucleotide sequences as flanking regions. These flanking regions provided suitable insertion sites within the chloroplast genome for cassette integration via homologous recombination. Simultaneous expression of the chloramphenicol‐resistant conferring gene and the gene coding for TurboGFP driven by 5′rbcL/5′atpB showed a potent natural bidirectional promoter as a reliable genetic tool.

中文翻译：

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叶绿体双重发芽启动子质粒，用于在Tetraselmis suecica（绿藻科，绿藻科）中表达异源蛋白质。

真核绿色微藻Tetraselmis suecica通常用于水产养殖，因为它具有较高的耐胁迫性，并且易于在多种环境中进行培养。因此，它们是生物技术应用的合适候选人。迄今为止，尚无有关基于特定内源启动子和同源靶向区域的叶绿体转化载体的数据。我们报告关于Tetraselmis suecica的鉴定基因编码核糖二磷酸羧化酶/加氧酶大亚基蛋白，光系统II D1蛋白和ATP合酶CF1-β亚基蛋白以及它们的非翻译区（5'UTR，3'UTR）。获得推定基因的全长ORF及其调控序列。我们还能够鉴定大亚基核糖体RNA基因（23S）的下游3'端以及与互补链上psb A基因首尾相连的5S RNA 。这些基因之间的基因间区域似乎是整合靶蛋白的良好靶位点。此外，我们在rbc L和atp之间鉴定了一个背对背启动子区域B基因。为了评估这两种启动子的双向性的活动，双报告载体构建了用于扁藻suecica含有转化猫由5'驱动和的TurboGFP基因RBC L / 5'三磷酸腺苷乙发散启动子。该载体包含23S-5S和psb A核苷酸序列作为侧翼区域。这些侧翼区域提供了叶绿体基因组内合适的插入位点，用于通过同源重组进行盒整合。通过5'驱动氯霉素抗性赋予基因和该基因编码的TurboGFP的同时表达红细胞L / 5' ATP乙表现出潜在的自然双向启动子作为一种可靠的基因工具。