Analysis of mechanism of pollen meiosis and its application to breeding.

在早熟期百合小孢子发生过程中检测到花粉母细胞蛋白（PMCP）。我们分析了PMCP在核小体核小体上的定位，在4-25%线性蔗糖梯度下，在4‰的温度下离心24h (25000 rps)，可以分离到二聚体和三聚体。各组均观察到组蛋白。SDS-PAGE法检测核小体单体中PMCP的含量。含有PMCP的单体可能在天然的5%聚乳酸凝胶电泳上延迟。因为核小体的高贵性取决于它们的分子大小、电荷等。我们用密度计测量了每个核小体单体、二聚体和三聚体的PMCP含量。以H4组蛋白含量计算，二聚体和三聚体的PMCP含量分别比单体高2倍和3倍。在本实验中，三聚体以上聚合物的PMCP含量几乎与三聚体相当。密度分析表明，PMCP定位于脂质体连接体结构域。PMCP可能在染色质高阶结构的组织中起着与HI组蛋白不同的作用。最后，我们尝试确定PMCP的部分氨基酸序列。采用高效液相色谱法分离PMCP和Hl，并应用于蛋白序列分析。但氨基酸信号未出现。这种失败是由于N端氨基酸的阻滞。然后我们检测了PMCP的切割。醋酸、n -溴代丁二酰亚胺、溴化氰等化学试剂可有效地将PMCP裂解为肽。

In microsporogenesis of lily PMCP(Pollen Mother Cell Protein) was detected after preneiosis stage. We analyzed PMCP localization on nucleosone Nucleosome sonomer,diner and trimer could be isolated with 4-25% linear sucrose gradient for 24hr centrifugation (25,000 rps) at 4ﾟC. Histones were observed in all fractions. PMCP was detected In nucleosome monomer by SDS-PAGE analysis. It is possible that monomer containing PMCP delayed on native 5% polyscrylaside gel electrophoresis. Because nobility of nucleosome depends on their molecular size,charge and so on.We measured PMCP content per nucleosome monomer,diner and trimer with densitometer.The PMCP contents of diner and trimer are two and three fold higher than that of monomer, respectively, as based on the contents of the H4 histone. PMCP contents of polymers more than trimer are nearly comparable to that of trimer in this experiment. From densitometrical analysis, it is suggested that PMCP localizes on nuoleosome linker domain. PMCP may play an important role for organization of high order structure of chromatin with another mode than HI histone.Finally, we tried to determine partial amino acid sequence of PMCP. PMCP and Hl were isolated by HPLC and applied to Protein sequense . However,amino acid signal could not be appeared. This failure is due to the blockins of N terminal amino acid. Then we examined cleavage of PMCP. Chemical reagent such as acetic acid, N-Bromosuccinicimidre, and cyanogen bromide were useful for cleavage of PMCP to peptide .