Wang T W, Balsamo R A, Ratnayake C, Platt K A, Ting J T, Huang A H
Department of Botany and plant Sciences, University of California, Riverside 92521, USA.
Plant J. 1997 Mar;11(3):475-87. doi: 10.1046/j.1365-313x.1997.11030475.x.
mRNAs encoding putative oleosins have been detected in the tapetum of developing anthers in Brassica and Arabidopsis, but the authentic proteins have not been previously documented. Antibodies against a synthetic 15-residue polypeptide that represents a portion of the putative tapetum oleosins encoded by two cloned Brassica napus genes were raised. Using these antibodies for immunoblotting after SDS-PAGE of the sporophytic extracts of B. napus developing anthers, two oleosins of approximately 48 and 45 kDa were detected. These two oleosins were judged to be the putative oleosins encoded by cloned Brassica genes because of their identical N-terminal sequences. The two oleosins were present in the anthers only during the developmental stage when the tapetum cells were packed with organelles. A fraction of lowdensity organelles was isolated from the developing anthers by flotation centrifugation. The fraction contained plastoglobule-filled plastids and lipid-containing particles. The structures of these two isolated organelles were similar to those in situ in the tapetum cells. Of subcellular fractions of the anther homogenate, the two oleosins were present exclusively in the low-density organelle fraction. They were absent in the surface fractions of the developing microspores and the mature pollen, although fragmented oleosin molecules were earlier reported to be present on the pollen. By immunocytochemistry, immunogold particles were found largely on the periphery of the plastoglobuli inside the plastids in the tapetum cells. The antibodies also detected oleosins on the surface of storage oil bodies inside the maturing microspores. Apparently, the gametophytic microspore oil-body oleosins share common epitopes at the generally non-conserved C-terminal domain with the sporophytic tapetum oleosins.
在甘蓝型油菜和拟南芥发育中的花药绒毡层中已检测到编码假定油质蛋白的信使核糖核酸（mRNAs），但此前尚未记录到真正的蛋白质。针对一种合成的15个氨基酸的多肽制备了抗体，该多肽代表由两个克隆的甘蓝型油菜基因编码的假定绒毡层油质蛋白的一部分。使用这些抗体对甘蓝型油菜发育中的花药的孢子体提取物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）后进行免疫印迹，检测到两种分子量约为48 kDa和45 kDa的油质蛋白。由于这两种油质蛋白具有相同的N端序列，因此被判定为由克隆的甘蓝型油菜基因编码的假定油质蛋白。这两种油质蛋白仅在绒毡层细胞充满细胞器的发育阶段存在于花药中。通过浮选离心从发育中的花药中分离出一部分低密度细胞器。该部分包含充满质体小球的质体和含脂颗粒。这两种分离的细胞器的结构与绒毡层细胞原位的结构相似。在花药匀浆的亚细胞组分中，这两种油质蛋白仅存在于低密度细胞器组分中。在发育中的小孢子和成熟花粉的表面组分中没有它们，尽管此前有报道称花粉上存在破碎的油质蛋白分子。通过免疫细胞化学方法，发现免疫金颗粒主要位于绒毡层细胞质体内质体小球的周边。这些抗体还在成熟小孢子内的贮藏油体表面检测到了油质蛋白。显然，配子体小孢子油体油质蛋白与孢子体绒毡层油质蛋白在通常非保守的C端结构域具有共同的表位。
