拟南芥菜(Arabidopsis thaliana)子叶中蛋白体形成的超微结构和免疫电镜定位研究

AN ULTRASTRUCTURAL AND IMMUNO-ELECTRON-MICROSCOPIC STUDY OF THE PROTEIN BODY FORMATION IN COTYLEDON CELLS OF ARABIDOPSIS THALIANA L.

本文对拟南芥菜(Arabidopsis thaliana)种子发育过程中贮藏蛋白的积累和蛋白体的形成进行了超微结构和免疫电镜定位的研究。常规超薄切片的电镜观察表明,在开花后第10天(10 DAF),高电子密度的蛋白质物质开始在子叶细胞的液泡中沉积。这一过程一直延续到种子接近成熟(14 DAF),这时液泡中充满了蛋白质物质,转变成为大的蛋白体。利用了该种植物主要种子贮藏蛋白之一的12 s球蛋白的单克隆抗体作为免疫探针,以蛋白质A-胶体金电镜技术对12 s种子蛋白进行了细胞内定位,证实了在液泡中积累的物质为种子贮藏蛋白。实验结果表明在拟南芥菜中,子叶细胞中的液泡是蛋白体的前体,肯定了蛋白体的发生起源于液泡的观点。本文还对应用胶体金电镜技术进行细胞内定位的某些问题作了初步探讨。

In higher plants,the seed storage pro-teins are deposited and sequestered in a spe-cial kind of organelle-the protein bodies.Thestructure,chemical composition and formationof this subcellular structure have been activetopics of research.On the other hand,somediscrepancies regarding the origin and onto-geny of protein bodies still remain.Resultsfrom different studies support one of the fol-lowing views:a) the vacuole origin,b) theER origin and c) the plastid origin. In this paper,the accumulation of seedstorage protein and the formation of proteinbodies in Arabidopsis thaliana have been stu-died ultrastructurally using the thin-section-ing and immuno-electronmicroscopy.The con-ventional thin sections viewed under theTEM showed that the electron-dense protein-aceous material started to deposit within thevacuoles in cotyledon cells 10 days after flo-wering (10 DAF).This process continued tothe near maturity of the seeds (14 DAF),atwhich time the large vacuoles were filledwith proteinaceous material and had turnedinto protein bodies.Meanwhile,monoclonalantibodies raised against the 12s globulin,one of the major storage proteins in thisspecies,were used as immuno-probes to loca-lize the antigen protein intracellularly throu-gh the protein A-gold TEM procedure.Inboth the resin embedded and freeze ultrathinsections processed with this technique,thecolloid gold particles (5nm mean diameter)were specifically localized within the electron-dense material accumulated inside the vacuo-les.This result confirmed that the electron-dense material was the seed storage protein,therefore the vacuoles represented the precur-sors of protein bodies.These data providedconvincing evidence that the protein bodies inthis plant have a vacuolar origin.Besides,some problems involved in the utilization ofcolloid gold TEM technique in intracellularimmuno-localization have been briefly discus-sed.