| 研究課題/領域番号 |
22K20651
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| 研究機関 | 名古屋大学 |
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研究代表者 |
Kozgunova Elena 名古屋大学, 高等研究院(理), 特任助教 (90786120)
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| 研究期間 (年度) |
2022-08-31 – 2025-03-31
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| キーワード | cell division / Physcomitrium patens / genetic screening / CRISPR/Cas9 |
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| 研究実績の概要 |
The core idea of this project is to combine CRISPR gene editing with the technological advantages of the model plant Physcomitrium patens (Physcomitrium patens) to achieve genetic screening with unprecedented speed and efficiency. This study focuses on genes that have not yet been functionally evaluated. We used transcriptome data from various cell cycles and developmental stages to create a library with genes that are thought to be involved in cell division. Then, about 800 genes were selected and genetic screening using CRISPR/Cas9 gene editing was carried out.
Based on the CRISPR/Cas9 screening in moss Physcomitrium patens, we identified three promising mutants, that are likely involved in the plant cell division and cytoskeleton organization. These genes have not been functionally analyzed previously, therefore we were able to identify novel players in plant cell division using CRISPR/Cas9 screening. Two of identified genes are conserved in bryophytes and ferns, but appear to be lost in angiosperms, suggesting that cell division machinery has evolved and lost certain components during plant evolution. Another gene is highly conserved across multiple plant species. Next, we plan to focus on detailed functional analysis of newly identified genes.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
At present, we are carrying out functional analysis of genes identified through CRISPR/Cas9 screening. We perform localization check by expressing respective protein fusion with GFP and observing where does protein localize inside the cell. We found that one of the proteins localize to the chromosomes during cell division, another one to the spindle and phragmoplast midzone. Due to certain problems, such as low level of protein expression, we so far were unable to observe localization of the third protein and are currently working to resolve this problem by overexpressing the protein. In addition , we perform detailed imaging analysis of mutants to gain insights into each gene function. We are also currently preparing manuscript for publication based on these data.
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| 今後の研究の推進方策 |
In future, we will further investigate protein functions. In addition to localization analysis and mutant phenotype, we plan to perform in vitro assays. By purifying the protein and testing its interaction with DNA or microtubules, we can gain better understanding of its function. We also aim to reveal protein-protein interactions through co-immunoprecipitation to isolate protein complexes, followed by mass-spectrometry to identify binding partners.
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| 次年度使用額が生じた理由 |
In terms of project budgeting, expenditures on cloning reagents, including restriction enzymes and DNA polymerases, turned out to be slightly lower than initially anticipated, largely owing to promotional offers provided by manufacturers at the time of procurement.Furthermore, the surplus from the previous budget cycle exceeded our initial projections, as some of the planned experiments were delayed and will be carried out this year. Surplus funds will be carried over and allocated towards purchasing consumables for this year experiments.
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