Modeling of Pigment Glycoside Production and Its Regulation by Genetic Transformation in Plant Cell Culture.

本研究旨在建立一种测定植物细胞培养过程中色素积累分布的新方法。根据这种分布的量化结果，建立了色素积累及其随培养周期变化的模型，用于细胞系的选择和色素产生途径的遗传转化。1)草莓细胞培养中花青素生产谱的定量分析：构建了一种利用图像分析定量测定每个细胞中色素积累谱的新方法，并验证了该方法的可重复性和实用性。2)通过遗传转化花青素形成途径提高草莓培养细胞色素产量：证实在培养基中添加苯丙氨酸作为前体，可以提高草莓细胞的花青素产量。由于前体进入细胞的摄取速率表明，花青素生产途径中的限速酶之一是CHS，因此试图通过将CHS基因引入草莓细胞的遗传转化来提高其产量。为了对花青素产生细胞系突变体进行遗传分析，采用连续培养的方法稳定了花青素产生途径的基因表达。采用连续培养法和cDNA减法对突变体中表达的特定基因进行了克隆研究。

The purpose of the study is to construct a novel method to determine the distribution of pigment accumulation among each cell in plant cell culture. As the results of the quantification of this distribution, a model describing pigment accumulation and its variation through culture period was made for the selection of cell lines and the genetic transformation in pigment production pathway.1) Quantification of an anthocyanin production profile in strawberry cell culture :A new method for the quantitative determination of pigment accumulation profiles in each cell using image analysis was constructed and verified as a reproducible and practical method.2) Genetic transformation of anthocyanin formation pathway for the enhancement of the pigment productivity in strawberry cultured cells :It was verified that the addition of phenylalanine as a precursor in the culture medium enhanced the anthocyanin productivity in strawberry cells. As the uptake rate of the precursor into the cells suggests that one of the rate-limiting enzyme in an anthocyanin production pathway is a CHS,the genetic transformation by the introduction of a CHS gene into the strawberry cells was tried to enhance the productivity. For the genetic analysis of a mutant in anthocyanin-producing cell lines, a continuous culture method was applied for the stabilization of gene expression in anthocyanin production pathway. Cloning of specific gene expressed in the mutant was investigated using a proposed method with a continuous culture and cDNA subtraction.