アブラナ科植物の受粉シグナル分子の解明

在进行正向遗传筛选后，我分离出授粉过程后具有缺陷的突变系。今年，我利用基因组深度测序确定了突变表型背后的致病基因。T-DNA插入株系的表型与遗传筛选株系的表型相似，表明该基因对拟南芥的受精过程具有重要作用。生物信息学分析表明，该基因与酿酒酵母（Saccharomycescerevisiae）胞外蛋白分选相关蛋白13（ScVPS 13）具有同源性，因此将该候选基因命名为AtVPS 13。vps 13成熟花粉处于三细胞期，花粉粒发育正常。因此，我观察了突变体花粉在柱头上的体内水合和萌发。我发现vps 13突变并不影响花粉的水合作用，然而，萌发严重受损（在授粉后45分钟内没有突变花粉萌发，但94%的野生型花粉萌发）。离体花粉萌发试验也显示出类似的趋势，表明VPS 13对花粉萌发很重要。结果，突变体植物的种子/角果的数量比野生型低几乎三倍。我还发现，除了vps 13之外，假定的paratinase AT 4G 17140的突变导致双突变体植物的严重不育表型。综上所述，我成功地鉴定了拟南芥成功受精的新基因，发现该基因在花粉水合后发挥作用，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.