Introduction The microalga Parachlorella kessleri-I produces high biomass and lipid content that could be suitable for producing economically viable biofuel at a commercial scale. Sequencing the complete chloroplast genome is crucial for the construction of a species-specific chloroplast transformation vector. Methods In this study, the complete chloroplast genome sequence (cpDNA) of P. kessleri-I was assembled; annotated and genetic transformation of the chloroplast was optimized. For the chloroplast transformation, we have tested two antibiotic resistance makers, aminoglycoside adenine transferase (aadA) gene and Sh-ble gene conferring resistance to spectinomycin and zeocin, respectively. Transgene integration and homoplasty determination were confirmed using PCR, Southern blot and Droplet Digital PCR. Results The chloroplast genome (109,642 bp) exhibited a quadripartite structure with two reverse repeat regions (IRA and IRB), a long single copy (LSC), and a small single copy (SSC) region. The genome encodes 116 genes, with 80 protein-coding genes, 32 tRNAs and 4 rRNAs. The cpDNA provided essential information like codons, UTRs and flank sequences for homologous recombination to make a species-specific vector that facilitated the transformation of P. kessleri-I chloroplast. The transgenic algal colonies were retrieved on a TAP medium containing 400 mg. L-1 spectinomycin, but no transgenic was recovered on the zeocin-supplemented medium. PCR and Southern blot analysis ascertained the transgene integration into the chloroplast genome, via homologous recombination. The chloroplast genome copy number in wildtype and transgenic P. kessleri-I was determined using Droplet Digital PCR. Conclusion The optimization of stable chloroplast transformation in marine alga P. kessleri-I should open a gateway for directly engineering the strain for carbon concentration mechanisms to fix more CO2, improving the photosynthetic efficiency and reducing the overall biofuels production cost.

中文翻译：

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叶绿体基因组的表征促进了 Parachlorella kessleri-I 的转化，这是一种用于生物燃料生产的潜在海藻

简介 微藻 Parachlorella kessleri-I 产生高生物量和脂质含量，可适用于以商业规模生产经济上可行的生物燃料。对完整的叶绿体基因组进行测序对于构建物种特异性叶绿体转化载体至关重要。方法在本研究中，组装了P. kessleri-I的完整叶绿体基因组序列（cpDNA）；优化了叶绿体的注释和遗传转化。对于叶绿体转化，我们测试了两种抗生素抗性标记物，氨基糖苷腺嘌呤转移酶 (aadA) 基因和 Sh-ble 基因，它们分别赋予了对壮观霉素和 zeocin 的抗性。使用 PCR、Southern 印迹和液滴数字 PCR 确认转基因整合和同型性测定。结果 叶绿体基因组 (109, 642 bp) 呈现出具有两个反向重复区域 (IRA 和 IRB)、一个长单拷贝 (LSC) 和一个小单拷贝 (SSC) 区域的四分结构。该基因组编码 116 个基因，其中 80 个蛋白质编码基因、32 个 tRNA 和 4 个 rRNA。cpDNA 为同源重组提供了重要信息，如密码子、UTR 和侧翼序列，以制造促进 P. kessleri-I 叶绿体转化的物种特异性载体。在含有 400 mg 的 TAP 培养基上回收转基因藻类菌落。L-1 大观霉素，但在添加 zeocin 的培养基上没有回收到转基因。PCR和Southern印迹分析通过同源重组确定了转基因整合到叶绿体基因组中。野生型和转基因 P. kessleri-I 中的叶绿体基因组拷贝数使用液滴数字 PCR 确定。