阿拉伯芥與馬拉巴栗葉肉細胞的葉綠素酶亞細胞定位

Abstract

葉綠素酶（clorophyllase, Chlase或CLH）是啟動葉綠素降解過程的主要酵素之一，能將葉綠素水解成脫植醇葉綠素（chlorophyllide）及植醇（phytol）。根據前人研究顯示，CLH位於柑橘的果皮色質體或銀杏葉和馬拉巴栗（Pachira aquatica）的葉綠體中；而在其他文獻指出，CLH不是位於葉綠體中，而在阿拉伯芥（Arabidopsis thaliana）中葉肉細胞的細胞質、液泡膜或內質網上。因此本研究將確定野生種阿拉伯芥及馬拉巴栗的CLH於不同發育時期中的表現模式。首先利用各種生物資訊資料庫分析比對馬拉巴栗葉綠素酶1（PaCLH1）與阿拉伯芥葉綠素酶（AtCLHs）的序列相似性及預測AtCLHs定位，結果顯示PaCLH1與AtCLHs有高序列相似性，而且預測CLHs是定位在細胞質和葉綠體。其次以Anti-PaCLH1抗體進行西方墨點法、免疫螢光染色和免疫金定位來分析兩物種CLH的亞細胞定位。西方墨點結果表明CLH蛋白表現量會隨著葉片的衰老程度而降低；在免疫細胞化學定位中，阿拉伯芥AtCLHs螢光訊號皆在葉綠體上；而CLH的免疫金標記定位在兩物種中的葉綠體、細胞質和液泡中，但不存在於線粒體和細胞壁上。 Chlorophyllase (Chlase or CLH), which hydrolyzes chlorophyll to form chlorophyllide and phytol, is one of the major enzymes to initiate the process of chlorophyll degradation pathway. It has been reported that CLH is localized in the peel plastid of citrus or the leaf chloroplast of Ginkgo biloba and Pachira aquatica. However, some literatures have suggested that CLH is not localized in the chloroplast, but is in the cytosol or tonoplast or ER of Arabidopsis thaliana mesophyll cells. In this study, the expression pattern of CLHs in the wild-type Arabidopsis thaliana and Pachira aquatica in different developmental stages would be verified. Sequence similarity and prediction of subcellular localization of Pachira aquatica chlorophyllase 1 (PaCLH1) and Arabidopsis thaliana chlorophyllases (AtCLHs) were first analyzed by various bioinformatics databases. The results demonstrate that PaCLH1 and AtCLHs share high sequence similarity. Besides, CLHs are preferentially predicted to localize in cytoplasm and chloroplast. Furthermore, anti-PaCLH1antibody was used for western blotting, immunocytochemistry (ICC) staining and immunogold labelling experiments to analyze the subcellular localization of CLH of the two species. The results show that protein level of CLH decreases during leaf aging. ICC data suggest that immunefluorescent signal of AtCLHs is localized to chloroplast. However, immunogold labeling results show that AtCLHs and PaCLH appeared in chloroplast, cytosol and vacuole but not in mitochondrium and cell wall.