七美絲瓜果實溢流42.5kDa蛋白質純化、鑑定與免疫定位

Abstract

在七美絲瓜（Luffa cylindrica Var. Seven Beauty）韌皮部溢流物中，經原態電泳分析蛋白質分子量分佈極廣，且有明顯10群主要蛋白質。利用修飾的雙向電泳進行占28.2％的85 kDa蛋白質純化。此主要蛋白質帶在第二方向變性膠體出現48、42.5、32、28、25蛋白質帶，其中以42.5 kDa為最主要蛋白質並且給予回收，回收率達15.98％。回收蛋白質經HPLC Reverse-phase Column分析在3.81分鐘的延遲時間22.86％的acetonitrile被沖洗為單一純化蛋白質。在西方墨漬法證實兩者免疫親緣關係，42.5kDa為85kDa蛋白質的次體。在42.5kDa蛋白質氨基酸成分以Glycine，Histidine為主要氨基酸共佔39.61％。另外，在經溴化氰斷裂後產生39.47、34.8、28.3、21.91、20.68kDa勝?片段，其中39.47kDa勝?的氨基端氨基酸序列為DTHFHVAVTTWTTK。28.3kDa勝?的氨基端氨基酸序列為TTGGDGGLGTKG。 在免疫螢光及免疫電顯定位下，85 kDa蛋白質只發現專一性分佈在維管束中老化的篩胞及導管細胞中。因此可知，在韌皮部溢流物的85kDa蛋白質為維管束專一性之晚期表現的蛋白質。

Phloem exudate obtained from Luffa cylindrica var. Seven Beauty fruit was separated with modified 2-D gel electrophoresis. A major protein of 85 kDa occupying about 28.2% of total proteins had 5 bands on denatured gel. One of major proteins at 42.5 kDa was further identified by Reversed - Phase HPLC showing one peak on 3.81 minute retention time and 22.86% acetonitrile. The cross reaction of the western blotting occurred on both 85 kDa and 42.5 kDa proteins, and it implied that 42.5 kDa was a subunit of 85 kDa protein. Glycine and Histidine that occupied 39.61% of total amino acid were major amino acid component of 42.5 kDa protein. The 42.5 kDa protein was digested into 20.68, 21.91, 31.96, 34.28 and 39.47 kDa peptide fragments by cyanogen bromide. The N-terminal amino acid sequence of 39.47 kDa is DTHFHVAVTTWTTK and 31.96 kDa is TTGGDGGLGTKG. Immunocytochemistry showed that 85 kDa protein only presented mature sieve element and vessel. It indicated that 85 kDa protein is specific for later expression in vascular bundle.