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

在百合小孢子发生过程中，花粉母细胞蛋白(PMCP)在受精卵形成后才出现。我们分析了PMCP在核小体单体上的定位，用4-25%的线性蔗糖梯度在4ﾟC下离心24小时(25,000 RPS)可以分离出DINER和TIMER。经SDS-PAGE分析，核小体单体中检测到PMCP。含有PMCP的单体可能延迟了5%的天然聚水杨酸乙酯凝胶电泳法。由于核小体的高贵程度取决于它们的分子大小、电荷等，因此我们用密度计测量了每个核小体单体、Diner和Trimer的PMCP含量，根据H4组蛋白的含量，Diner和Trimer的PMCP含量分别是单体的2倍和3倍。在本实验中，三聚体以上的聚合物的PMCP含量与三聚体的含量几乎相当。密度计量学分析表明，PMCP定位于核小体连接区。PMCP可能以另一种方式参与染色质高阶结构的组织，而不是HI组蛋白。最后，我们尝试测定了PMCP的部分氨基酸序列。用高效液相色谱分离PMCP和HL，并用于蛋白质序列测定。然而，氨基酸信号不能出现。这一失败是由于N末端氨基酸的阻塞。然后我们检测PMCP的裂解情况。化学试剂如冰醋酸、N-溴代丁二酰亚胺、溴化氰等有助于将PMCP裂解成多肽。

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 .