阿拉伯芥中Osmotin同源蛋白質之細胞定位與突變株表型之分析

Abstract

菸草的Osmotin蛋白質會透過酵母菌細胞膜表面的接受體YOL002c，誘發酵母菌進行細胞凋亡。阿拉伯芥之HHP1 (heptahelical protein 1) 與YOL002c有高度序列保守性 (30% identity)，皆為預測具有七個穿膜區塊的膜蛋白質。在過量表現osmotin的橄欖樹轉殖株中發現，較野生型有較多進行細胞凋亡的細胞。本篇研究目的在研究阿拉伯芥中的osmotin同源蛋白質AtOSM34與AtOLP在植物體的功能。利用pHBT95sGFP表現載體建構含 osmotin之綠色螢光融合蛋白質，於洋蔥表皮細胞與阿拉伯芥原生質體進行短暫表現，發現AtOSM34與AtOLP可能位於細胞質中。本研究建立AtOLP突變株，進一步探討其表現型是否與逆境相關。進行鹽逆境與水逆境處理後，發現AtOLP突變株在較高濃度鹽逆境與滲透壓逆境下，發芽率較野生型差，推論AtOLP可能與此逆境相關。本研究企圖建立以流式細胞儀觀察細胞週期，外加Osmotin處理阿拉伯芥原生質體，觀察是否誘發細胞凋亡。

Osmotin from tobacco can induce apoptosis of yeast by binding to a yeast membrane protein, YOL002c. HHP1 (heptahelical protein 1) is a homolog (30% sequence identity) of YOL002c in Arabidopsis. Both HHP1 and YOL002c are predicted to have a seven-transmembrane domain. We were interested in whether or not osmotin can interact with HHP1 in Arabidopsis. AtOSM34 and AtOLP are Arabidopsis proteins homologous to osmotin from tobacco. Transient expression of AtOSM34-sGFP and AtOLP-sGFP fusion proteins in onion epidermal cells and Arabidopsis protoplasts showed that osmotin are most likely to be located in cytosol. Heterologous protein expression of AtOSM34 and AtOLP in E. coli was established. T-DNA knock-down mutant of AtOLP was established using an ABRC line (WiscDsLox). We found that the mutant showed a slight decreasing in germination rate in high concentration of NaCl or mannitol. To determine whether or not osmotin could interact with HHP1, we established an apoptosis analysis method using flow cytometry with isolated nuclei.