アブラナ科植物の受粉シグナル分子の解明

进行正向遗传筛选后，经授粉过程筛选出缺陷突变株系。在这一年里，我用基因组深度测序确定了突变表型背后的致病基因。鉴定基因的T-DNA插入株系表现出与遗传筛选分离的T-DNA插入株系相似的表型，表明鉴定的基因对拟南芥受精具有重要意义。生物信息学分析表明，该基因可能与酿酒酵母空泡蛋白分选相关13(ScVPS13)基因同源，因此本研究将该候选基因命名为AtVPS13。Vps 13的成熟花粉处于三细胞期，花粉粒发育正常。因此，我在活体上观察到了柱头上突变花粉的水合和萌发。我发现vps13突变不影响花粉的水合作用，但严重影响了花粉的萌发(授粉后45min内没有突变花粉萌发，但94%的野生型花粉萌发)。花粉体外萌发试验也表现出类似的趋势，表明VPS13对花粉萌发具有重要作用。结果表明，突变株的种子/角果数几乎是野生型的3倍。我还发现，除了vps13之外，假定的paralog AT4G17140突变会导致双重突变植物的严重不育表型。综上所述，我成功地鉴定了与拟南芥受精成功有关的新基因，发现该基因在花粉水合后起作用，支持花粉在柱头上萌发。

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.