| 研究実績の概要 |
After forward genetic screen was performed, I isolated mutant lines with defect after pollination process. In this year, I identified the causal gene underlying the mutant phenotype using genome deep sequencing. T-DNA insertion plant line of the identified gene showed similar phenotype as the one isolated from genetic screen suggesting that the identified gene was important for fertilization of Arabidopsis. Bioinformatic analysis revealed that this gene is a putative homolog of Saccharomyces cerevisiae Vacuolar Protein-Sorting associated 13 (ScVPS13), so, the candidate gene was named AtVPS13 in this study. Pollen grain development of vps13 was confirmed to be normal since its mature pollen was in tricellular stage. So, I observed in vivo hydration and germination of the mutant pollen on the stigma. I found that vps13 mutation did not affect hydration of the pollen however, the germination was severely compromised (no mutant pollen germinated within 45 min after pollination, but 94% of wild type pollen germinated). In vitro pollen germination assay also showed similar trend suggesting that VPS13 was important for pollen germination. As a result, the number of seed/silique of mutant plant was almost three times lower than the wild type. I also found that mutation of putative paralog AT4G17140 additional to vps13 results in a severe sterile phenotype of double mutant plant. In summary, I successfully identified novel gene important in successful fertilization of Arabidopsis found that this gene functioned after pollen hydration to support pollen germination on the stigma.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
The project has progressed in this year more than the initial plan. Apart from confirming the identity of causal gene responsible for mutant phenotype, further analyses were done to pin point the step this gene was involved in the fertilization process of Arabidopsis. Also, the identified gene was a novel gene which no one has ever studied in Arabidopsis before. At this point, it is very interesting to see more specific role of this gene in pollen germination.
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| 今後の研究の推進方策 |
At this point, I am getting the idea of which stage after pollination required the function of identified gene. I will show the importance of this gene in the fertilization of Arabidopsis by using transmission efficiency assay to compare the ability of mutant and wild-type pollen grains. The homozygous plant having one mutant allele of the gene of interest will be used to perform reciprocal cross with wild-type plant. I also plan to generate transcriptional fusion of the promoter of the gene of interest and beta-D-glucuronidase reporter gene. After introducing the construct into Arabidopsis, the reporter gene activity will be observed to show the organs/tissues which the gene of interest is being transcribed. The study of protein sequence suggested that this gene might involve in calcium signaling in the pollen. I plan to observe the calcium dynamic in pollen grain by expressing calcium-sensor fusion protein in the transgenic plant having mutant allele of the gene of interest, to be compared with the transgenic plant carrying wild-type allele. The reporter pollen will be used in a live-imaging experiment to reflect calcium dynamic of the wild-type and mutant pollen grain after the attachment on the stigmatic papilla cell. Microscopic observation using transmission electron microscopy is also planned. By observing wild-type and mutant pollen grain after pollination at the organelle resolution, I should be able to see cause of failure for the mutant pollen and get the idea of cellular process which is dependent on the function of the gene of interest.
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