Hormone-induced differentiation of BY-2 cultured tobacco cells ; development of a model system for differentiation of root-cap cells.

在含有生长素(2，4-D，0.2mg1；-1)的常规培养基中，BY-2培养的烟草细胞增殖活跃，形成由8~16个串联的小圆柱形细胞组成的简单细胞团，细胞内的质体在整个培养过程中保持未分化。相比之下，在含有细胞分裂素(BA)而不是生长素的改良培养基中，BY-2细胞变大，但没有活跃的细胞增殖，细胞内的质体分化为淀粉体。除了这些特性(不活跃的细胞增殖、细胞增大、淀粉体发育)外，我们还发现BY-2细胞在生长素耗竭的培养液中的行为类似于根冠细胞：(I)它们容易彼此剥落，(Ii)表现出较短的寿命，(Iii)表现出较高的分泌活性。检测到4种根冠细胞样BY-2细胞特异分泌的蛋白质，对纯化的蛋白质进行N端肽序列分析，确定其中一种为分泌型过氧化物酶SPCT44。对于其他三种蛋白质，由于N末端被封闭，我们无法确定N末端的多肽序列。通过测定内部肽序列来鉴定它们现在正在进行中。为了分析正常BY-2细胞向根帽状细胞分化过程中基因表达的变化，我们利用常规和改良培养12h的BY-2细胞中纯化的mRNA行ddRT-PCR和微阵列分析(与RIKEN合作)，鉴定了几个根据根帽状细胞分化而激活表达的基因。结果由-2\f25 RIKEN-2(http://mrg.psc.riken.go.jp/strc/).)在-2\f25 BY-2\f25 EST-2数据库中打开利用本研究获得的分子标记，进一步分析改良培养基中根冠细胞与类似根冠细胞的BY-2细胞的相似性。

In conventional medium containing auxin (2,4-D, 0.2mg1^<-1>), BY-2 cultured tobacco cells proliferate actively, forming simple cell clusters composed of 8 to 16 small cylindrical cells connected in tandem, and the plastids within the cells remain undifferentiated throughout the culture period. By contrast, in a modified medium that contain cytokinin (BA, 1mg1^<-1>) instead of auxin, BY-2 cells become large without active cell proliferation, and the plastids within the cells differentiate into amyloplasts. In addition to these characteristics (inactive cell proliferation, cell enlargement, amyloplast development), we found that the behavior of BY-2 cells in the auxin-depleted medium resembled to that of root-cap cells in that (i) they easily exfoliate with each other, (ii) exhibited short lifetime, and (iii) showed higher secretion activity. Four proteins specifically secreted by root-cap cell-like BY-2 cells were detected, and one of them (named SPCT44) was identified as secreted peroxidase by N-terminal peptide sequencing of the purified protein. For other three proteins, we could not determine N-terminal peptide sequence because N-terminal was blocked. Their identification by determining internal peptide sequence is now on progress. To analyze changes in the gene expression during differentiation of normal BY-2 cells to root-cap like ones, we performed ddRT-PCR and microarray analyses (collaboration with RIKEN), using mRNAs purified from BY-2 cells cultured 12 h in either conventional medium or modified medium, and identified several genes whose expression is activated according to differentiation of root-cap like cells. The results are opened in the BY-2 EST database by RIKEN (http://mrg.psc.riken.go.jp/strc/). I'd like to further analyze the similarity between root-cap cells and the root-cap cell-like BY-2 cells in the modified medium, by using molecular markers obtained through this research project.