| 摘要: | 本實驗室先前從甘薯老化葉片分離一個全長papain-like cysteine protease SPCP1 cDNA序列,其ORF對應至成熟的蛋白質部分已被構築表現融合蛋白及產生多株抗體。本研究利用該多株抗體及西方墨點雜交方法來探討甘藷 SPCP1基因的表現及調控。甘藷葉片在自然老化或黑暗、ethephon、ABA處理誘導老化過程皆會顯著增加SPCP1的表現量，SPCP1的表現具有組織專一性且顯著累積於老化的葉片上，然而於根、莖及年青葉片則無顯著表現。關於SPCP1基因表現時相關調控因子方面的研究結果顯示ethephon誘導SPCP1表現明顯的受還原態的glutathione (GSH) 及EGTA 所抑制，L-Buthionine-sulfoximine (BSO; glutathione 合成抑制劑)及calcium ionophore A23187，兩者分別單獨處理亦可誘導SPCP1的表現，此誘導作用在GSH及EGTA分別存在下會受到抑制。另外ethephon誘導SPCP1的表現在真核生物蛋白質抑制劑 cycloheximide (CHX) 存在下亦會受到抑制，其抑制時間約在ethephon處理後 3至6小時內。這些數據建議我們甘藷SPCP1是一個老化相關基因而且在甘藷葉片自然老化或黑暗、ethephon、ABA處理誘導老化過程被表現。另外ethephon 在誘導SPCP1基因表現過程的訊息傳導途徑中，Ca2+ 離子及活化氧族 (reactive oxygen species) 似乎扮演二次訊息 (secondary messenger) 的角色，而且也需要新合成的蛋白質。
We have previously isolated a full-length senescence-associated papain-like cysteine protease cDNA, SPCP1, in our laboratory. Its open reading frame encoding mature protein portion has been constructed and expressed as fusion protein in E. coli for polyclonal antibody production. In this study, the produced rabbit anti-SPCP1 polyclonal antibody was used for Western blot hybridization in order to explore the expression and regulation of SPCP1 gene. The expression of SPCP1 was enhanced in nature senescent leaves and dark-, ethephon-, or ABA-induced senescent leaves. SPCP1 exhibited tissue-specific expression and accumulated significant amount in senescing leaves, but not in young mature leaves, roots or stems. For the study of factors regulating SPCP1 expression, ethephon-induced SPCP1 expression was significantly repressed by reduced glutathione or EGTA. L-Buthionine-sulfoximine (BSO; an endogenous glutathione synthesis inhibitor) and calcium ionophore A23187 can separately induced SPCP1 expression, and the induction was also repressed by reduced glutathione and EGTA, respectively. In addition, cycloheximide (CHX; an inhibitor for eukaryotic protein de novo synthesis) also drastically reduced ethephon-induced SPCP1 expression and the time for significant repression was about between 3 and 6 hours after ethephon treatment. Based on these results sweet potato cysteine protease SPCP1 is a senescence-associated gene, and can be expressed in natural senescent leaves and dark-, ethephon- or ABA-induced senescent leaves. In addition, external calcium ion and reactive oxygen species may play roles as the possible secondary messengers for the signaling of ethephon induction of SPCP1 gene expression. De novo protein synthesis is also likely required. |
