Igawa Tomoko, Yamada Lixy, Sawada Hitoshi, Mori Toshiyuki
Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan.
Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Sugashima, Toba, Mie 517-0004, Japan.
Plant Biotechnol (Tokyo). 2017;34(2):119-123. doi: 10.5511/plantbiotechnology.17.0522a. Epub 2017 Jun 27.
Angiosperms possess a double fertilization system for sexual reproduction. Double fertilization is regulated by interactions among proteins localized in the plasma membrane of each sex gamete. A few plasma membrane resident proteins regulating double fertilization have been identified in male gametes. In contrast, no fertilization regulators in female gamete plasma membrane have been identified, largely due to difficulties in the isolation and collection of female gametes. We had produced Arabidopsis transgenic plant pDD45::GFP-AtPIP2;1 where the egg cell plasma membrane was specifically labeled with GFP (Igawa et al. 2013). The protein extract derived from approximately 200 pistils, which contained unfertilized and mature egg cells, was subjected to immunoprecipitation using anti-GFP antibody. As a result, both GFP and AtPIP2;1 were specifically detected in immunoprecipitated proteins from pistil tissues of pDD45::GFP-AtPIP2;1 transgenic plant, but not in those of wild type pistils. It was revealed that specific proteins expressed in the egg cells were successfully isolated from pistil cell population. The method described here showed the feasibility of isolating specific egg cell plasma membrane protein without gamete isolation and collection procedures.
被子植物拥有用于有性生殖的双受精系统。双受精受位于每个性配子质膜上的蛋白质之间的相互作用调控。在雄配子中已鉴定出一些调控双受精的质膜驻留蛋白。相比之下，尚未在雌配子质膜中鉴定出受精调节因子，这主要是由于雌配子的分离和收集存在困难。我们构建了拟南芥转基因植株pDD45::GFP-AtPIP2;1，其中卵细胞的质膜被GFP特异性标记（Igawa等人，2013年）。从大约200个雌蕊中提取的蛋白质提取物，其中包含未受精的成熟卵细胞，使用抗GFP抗体进行免疫沉淀。结果，在pDD45::GFP-AtPIP2;1转基因植株雌蕊组织的免疫沉淀蛋白中特异性检测到了GFP和AtPIP2;1，但在野生型雌蕊的免疫沉淀蛋白中未检测到。结果表明，成功地从雌蕊细胞群体中分离出了卵细胞中表达的特异性蛋白质。本文所述方法表明，无需进行配子分离和收集程序即可分离特定卵细胞质膜蛋白的可行性。
