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

BY-2 培养烟草细胞的激素诱导分化；

基本信息

批准号：
12640634
负责人：
SAKAI Atsushi
金额：
$ 3.07万
依托单位：
Nara Women's University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12640634/
关键词：
Auxin Cytokinin Root cap Tobacco BY-2 Secreted protein Microarray Peroxidase 培養細胞

项目摘要

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.

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至16个小圆柱细胞串联组成的简单细胞簇，其内的质体细胞在整个培养期间保持未分化。相反，在含有细胞分裂素(BA，1mg1^-1)而不是生长素的改良培养基中，BY-2细胞变大而没有活跃的细胞增殖，并且细胞内的质体分化成淀粉体。除了这些特征（细胞增殖不活跃、细胞增大、造粉体发育）之外，我们发现 BY-2 细胞在生长素耗尽的培养基中的行为与根冠细胞的行为相似，因为 (i) 它们很容易用彼此，（ii）表现出较短的寿命，（iii）表现出较高的分泌活性。检测到4种根帽细胞样BY-2细胞特异分泌的蛋白，通过纯化蛋白的N端肽测序，鉴定其中一种蛋白（命名为SPCT44）为分泌型过氧化物酶。对于其他三种蛋白质，我们无法确定 N 端肽序列，因为 N 端被封闭。通过确定内部肽序列对其进行鉴定目前正在进行中。为了分析正常 BY-2 细胞向根帽样细胞分化过程中基因表达的变化，我们使用从在传统培养箱中培养 12 小时的 BY-2 细胞中纯化的 mRNA 进行了 ddRT-PCR 和微阵列分析（与 RIKEN 合作）。培养基或改良培养基，并鉴定了一些基因，其表达根据根帽样细胞的分化而被激活。结果由 RIKEN (http://mrg.psc.riken.go.jp/strc/) 在 BY-2 EST 数据库中打开。我想通过使用通过本研究项目获得的分子标记来进一步分析改良培养基中根冠细胞和类根冠细胞BY-2细胞之间的相似性。