Metabolic research for development of opium poppy producing high amount of morphine

为了开发高产量吗啡的罂粟，本研究试图研究罂粟中吗啡的代谢机制，并克隆吗啡代谢酶基因。因此，我证明，在响应创伤，吗啡代谢成双吗啡的细胞壁过氧化物酶。在使用各种色谱法纯化这些酶之后，通过氨基加成测序仪分析纯化的酶，但是我不能获得关于酶的一级结构的信息。通过PCR反应获得吗啡代谢酶的克隆。第一步，利用植物过氧化物酶保守区设计的引物进行简并PCR。最后，通过5 '-RACE和3'-RACE分离了三个编码全长区域的基因。氨基酸序列分析表明，这些基因编码过氧化物酶。虽然用E. coil的时候，我不能以活性形式表达这些酶。现在我尝试用昆虫细胞来表达这些基因。

To develop opium poppy producing high amount of morphine, I attempted to investigate the metabolic mechanism of morphine in opium poppy and clone genes encoding morphine-metabolizing enzymes. Consequently, I demonstrated that in response to wounding, morphine is metabolized into bismorphine by cell wall peroxidases. After purification of these enzymes using various chromatography, the purified enzymes were analyzed by the amino-add sequencer, but I could obtain no information on the primary structures of the enzymes. The cloning of morphine-metabolizing enzymes was achieved by PCR reaction. As a first step, degenerate PCR was camed out using primers designed from the conserved regions in the plant peroxidases. Finally, three genes encoding full-length regions were isolated by 5'-RACE and 3'-RACE. Analysis of their amino add sequences demonstrated that these genes encode peroxidases. Although heterogeneous expression of them using E. coil was attempted, I could not express these enzymes in active forms. Now I try to express these genes using insect cells.