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

花粉减数分裂机制分析及其在育种中的应用

基本信息

批准号：
63490004
负责人：
HARADA Hiroshi
金额：
$ 2.11万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

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 .

在百合小孢子发生过程中，花粉母细胞蛋白（PMCP）在花粉母细胞分化前期以后被检测到。我们分析了PMCP在核小体上的定位。核小体的核糖体、二聚体和三聚体可以用4-25%线性蔗糖梯度在4 ℃离心24小时（25，000 rps）分离。在所有组分中均观察到组蛋白。SDS-PAGE分析显示PMCP存在于核小体单体中。含有PMCP的单体可能在天然的5%聚乙烯醇糖苷凝胶电泳上延迟。由于核小体的高贵性取决于核小体分子的大小、电荷等，我们用光密度计测定了核小体单体、二聚体和三聚体的PMCP含量，以H4组蛋白含量为基准，二聚体和三聚体的PMCP含量分别是单体的2倍和3倍。三聚体以上聚合物的PMCP含量与三聚体的PMCP含量基本相当。光密度分析表明，PMCP定位于核小体连接结构域。PMCP可能以不同于HI组蛋白的另一种方式对染色质的高级结构的组织起重要作用。通过HPLC分离PMCP和H1，并用于蛋白质测序。但未出现氨基酸信号。这一失败是由于N端氨基酸的封闭。然后我们检查了PMCP的切割。化学试剂如乙酸、N-溴代琥珀酰亚胺和溴化氰可用于将PMCP切割成肽。