兰州百合精细胞特异蛋白的研究

通过低渗冲击及Percoll密度梯度离心的方法,成功地分离并纯化了兰州百合(Lilium davidiiDuch.)生活的生殖细胞及精细胞。从精细胞、生殖细胞及叶片中提取了全蛋白,并通过双向电泳技术对它们进行了比较。在双向电泳图谱上精细胞比生殖细胞显示更多的蛋白斑点,特别是在碱性端。通过混合酶解及离心,分离了生活的叶肉原生质体。用生物素的琥珀酰胺酯衍生物(NHS-biotin)对精细胞、生殖细胞及完整的叶肉原生质体质膜蛋白进行标记,然后进行Western blot分析,用辣根过氧化物酶酶标链霉抗生物素蛋白及其底物4-氯-1-萘酚反应显色,比较了3种质膜蛋白。发现分子量为46kD及50kD的两种蛋白是精细胞质膜特异的。在双向电泳图谱上也可找到与这两种蛋白相对应的斑点,它们很可能与受精过程中精卵的识别有关。

THE SPECIFIC PROTEINS OF SPERM CELLS OF LILIUM DAVIDII

Viable generative cells and sperm cells of Lilium davidii Duch. were successfully isolated and purified with osmotic shock method and Percoll density gradient centrifugation. Total proteins were extracted from sperm cells, generative cells and leaves of L. davidii. Protein patterns were compared on two-dimensional-PAGE. There were apparently more protein dots on the gel from sperm cells than on that from generative cells, especially in the alkaline region. Viable leaf protoplasts were isolated with multi-enzyme mixture, followed by centrifugation. The plasma membrane proteins of sperm cells, generative cells and intact leaf protoplasts were compared, using NHS-biotin (N-hy-droxysuccinimido-biotin) labeling method. Horseradish peroxidase labeled streptavidin and the substrate 4-Chloro-l-naphthol were used to detect membrane proteins during Western blotting. Two proteins with molecular weights of 46 kD and 50 kD were specific to the plasma membrane of sperm cells. These two sperm specific protein dots were also found on the two-dimensional gels. These proteins may play key roles in sperm-egg recognition in the fertilization process.