七美絲瓜果實之24kDa溢流蛋白的分離、純化與定序

Abstract

植物韌皮部存在著一群複雜具組織專一性的蛋白質，唯至今對P-protein生化功能的瞭解仍很有限。本實驗室於前年從絲瓜果實的韌皮部溢流液中得到24kDa蛋白質的抗體，本實驗則分別利用SDS-PAGE及雙向電泳，對七美絲瓜果實的韌皮部溢流液及中長絲瓜的植株進行蛋白質之分離及免疫轉印分析，同時將其中的24kDa溢流蛋白純化，進行胺基酸組成分析及N-端序列定序。在SDS-PAGE電泳圖譜上，七美絲瓜果實溢流蛋白主要可以分為4群，而藉由免疫轉印的分析得知七美絲瓜的24kDa溢流蛋白可被抗體所辨識，除了24kDa的主帶外，還有少許其他蛋白質被辨識。在雙向電泳圖譜中，絲瓜果實溢流液以酸性蛋白居多，而在免疫墨漬分析上，可辨識一群分子量為24kDa，pI為4-5.2的蛋白（約有4-6個點）。此外，24kDa蛋白群在三天大的小苗中即被表現，並且存在木質部中。將其中一個分子量24kDa，pI 5.2的溢流蛋白純化，經胺基酸組成分析得知其組成以Glycine、Aspartic acid及Glutamic acid居多，分別占12.3％、8.9％及7.2％，對此蛋白質進行CNBr的斷裂反應，而後送至中部貴儀中心進行N-端序列分析。

P-proteins are groups of protein expressed specifically in the phloem. Their biological function have been not well defined so far. A polyclonal antibody against 24 kDa phloem protein was prepared in our previous study. In the present study, phloem proteins from Luffa cylindrica were separated with SDS-PAGE and 2-D electrophoresis. There are four groups of phloem protein in SDS-PAGE profile. A major band at 24 kDa and some minor bands can be identified by the polyclonal antibody. Most fruit phloem exudate protein are acid in 2-D gel. Immunoblotting study showed that 4-6 spots at 24 kDa with pI ranging from 4 to 5.2 are detected. The antibody of pepo 24 kDa also recognized the similar proteins expressed in a three-day-old seedling and in xylem sap. A 24 kDa protein with pI 5.2 obtained from 2-D gel have 16 kinds of amino acids with high contain of Glycine (12.3%) Aspartic acid (8.9%) and Glutamic acid (7.2%). The N-terminal sequencing is totally blocked. Further cleavage of 24 kDa by CNBr was tested and peptide fragments were transferred to PVDF membrane for sequence analysis.