Diplonemids are a group of highly diverse and abundant marine microeukaryotes that belong to the phylum Euglenozoa and form a sister clade to the well‐studied, mostly parasitic kinetoplastids. Very little is known about the biology of diplonemids, as few species have been formally described and just one, Diplonema papillatum, has been studied to a decent extent at the molecular level. Following up on our previous results showing stable but random integration of delivered extraneous DNA, we demonstrate here homologous recombination in D. papillatum. Targeting various constructs to the intended position in the nuclear genome was successful when 5′ and 3′ homologous regions longer than 1 kbp were used, achieving N‐terminal tagging with mCherry and gene replacement of α‐ and β‐tubulins. For more convenient genetic manipulation, we designed a modular plasmid, pDP002, which bears a protein‐A tag and used it to generate and express a C‐terminally tagged mitoribosomal protein. Lastly, we developed an improved transformation protocol for broader applicability across laboratories. Our robust methodology allows the replacement, integration as well as endogenous tagging of D. papillatum genes, thus opening the door to functional studies in this species and establishing a basic toolkit for reverse genetics of diplonemids in general.

中文翻译：

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通过同源重组的靶向整合能够在海洋微真核生物乳头状瘤中对基因进行原位标记和替换。

Diplonemids是一组高度多样化和丰富的海洋微真核生物，属于真核动物门，并与经过深入研究的寄生虫动植物体形成了姐妹进化枝。关于双倍体动物的生物学知之甚少，因为正式描述的物种很少，而在分子水平上仅对一种物种的乳头状双歧杆菌（Diplonema papillatum）进行了研究。遵循我们先前的结果，该结果显示了递送的外源DNA的稳定但随机整合，我们在此处证明了乳头状果蝇中的同源重组。当使用长于1 kbp的5'和3'同源区域时，将各种构建体靶向核基因组中的预期位置是成功的，从而实现了mCherry的N末端标记以及α-和β-微管蛋白的基因置换。为了更方便地进行基因操作，我们设计了带有p-A002的模块质粒pDP002，该质粒带有A蛋白标记，并用于生成和表达C末端标记的线粒体蛋白。最后，我们开发了一种改进的转换协议，可在各个实验室中更广泛地应用。我们可靠的方法可以对乳头状果蝇基因进行替换，整合和内源标记，从而为该物种的功能研究打开了大门，并为二倍体类动物的反向遗传学建立了基本的工具包。