Presentation Information
[P04-553]Establishment of genome resources and genetic transformation system in the marine diatom Navicula pelliculosa for ARS identification
○Miyu Iizuka1, Ryota Kumakubo1, Yoshiaki Maeda2, Kosuke Kataoka1,3, Tsuyoshi Tanaka1 (1. Tokyo University of Agriculture and Technology (Japan), 2. Institute of Life and Environmental Sciences, University of Tsukuba (Japan), 3. Comprehensive Research Organization, Waseda University (Japan))
Keywords:
Diatom,Bacterial conjugation,Episomal vector
[Purpose]
Diatoms are promising platforms for sustainable bioproduction from CO2. Molecular breeding based on metabolic engineering has been actively promoted in diatoms. However, introduction of large DNA constructs remains technically challenging. Although episomal vectors enable the delivery of long DNA sequences, most conventional systems rely on yeast-derived autonomously replicating sequence (ARS) elements, which show limited stability in diatom cells. The identification of diatom-derived ARS sequences is expected to improve episomal maintenance and expand genetic engineering applications. However, ARS information and comprehensive genomic resources remain limited in many diatom species. In this study, we aimed to establish fundamental technologies, including an efficient genetic transformation system and genome-based molecular resources, in the marine diatom Navicula pelliculosa as a basis for ARS identification.
[Method]
A genetic transformation system was first established in N. pelliculosa using plasmids carrying the neomycin resistance gene (nptII) introduced by particle bombardment. Tungsten particle size and bombardment pressure were optimized. Draft genome data were analyzed using Helixer (ver. 0.3.3), followed by homology-based identification of genes encoding the subunits of origin recognition complex (ORC). These subunits have been used as ChIP targets in Phaeodactylum tricornutum and are known to be associated directly with DNA within the ORC. FLAG tag-fused ORC expression plasmids were constructed and introduced into N. pelliculosa, and RT-PCR confirmed transcription of the tagged genes.
[Results&Consideration]
Optimization of particle bombardment yielded a maximum transformation efficiency of 430 clones per 10^8 cells (1.0 µm tungsten particles, 1,100 psi), exceeding that reported for Fistulifera solaris. Draft genome analysis revealed a genome size of 27.8 Mb (GC content 47%) with 13,568 predicted protein-coding genes. Four ORC homologs corresponding to ORC1, ORC2, ORC4, and ORC5 were identified, whereas ORC3 and ORC6 were not clearly detected under the current annotation conditions. The presence of multiple ORC core subunits suggests conservation of the replication initiation machinery in this species and supports feasibility of ORC-mediated chromatin analysis for ARS identification.
[Conclusion]
This study established essential technical foundations, including an efficient genetic transformation system and genome-based identification of ORC genes in N. pelliculosa. These results provide an experimental platform for ChIP-seq analysis aimed at identifying ARS genomic regions.
Diatoms are promising platforms for sustainable bioproduction from CO2. Molecular breeding based on metabolic engineering has been actively promoted in diatoms. However, introduction of large DNA constructs remains technically challenging. Although episomal vectors enable the delivery of long DNA sequences, most conventional systems rely on yeast-derived autonomously replicating sequence (ARS) elements, which show limited stability in diatom cells. The identification of diatom-derived ARS sequences is expected to improve episomal maintenance and expand genetic engineering applications. However, ARS information and comprehensive genomic resources remain limited in many diatom species. In this study, we aimed to establish fundamental technologies, including an efficient genetic transformation system and genome-based molecular resources, in the marine diatom Navicula pelliculosa as a basis for ARS identification.
[Method]
A genetic transformation system was first established in N. pelliculosa using plasmids carrying the neomycin resistance gene (nptII) introduced by particle bombardment. Tungsten particle size and bombardment pressure were optimized. Draft genome data were analyzed using Helixer (ver. 0.3.3), followed by homology-based identification of genes encoding the subunits of origin recognition complex (ORC). These subunits have been used as ChIP targets in Phaeodactylum tricornutum and are known to be associated directly with DNA within the ORC. FLAG tag-fused ORC expression plasmids were constructed and introduced into N. pelliculosa, and RT-PCR confirmed transcription of the tagged genes.
[Results&Consideration]
Optimization of particle bombardment yielded a maximum transformation efficiency of 430 clones per 10^8 cells (1.0 µm tungsten particles, 1,100 psi), exceeding that reported for Fistulifera solaris. Draft genome analysis revealed a genome size of 27.8 Mb (GC content 47%) with 13,568 predicted protein-coding genes. Four ORC homologs corresponding to ORC1, ORC2, ORC4, and ORC5 were identified, whereas ORC3 and ORC6 were not clearly detected under the current annotation conditions. The presence of multiple ORC core subunits suggests conservation of the replication initiation machinery in this species and supports feasibility of ORC-mediated chromatin analysis for ARS identification.
[Conclusion]
This study established essential technical foundations, including an efficient genetic transformation system and genome-based identification of ORC genes in N. pelliculosa. These results provide an experimental platform for ChIP-seq analysis aimed at identifying ARS genomic regions.
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