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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.

BY-2培养的烟草细胞在含有生长素（2，4-D，0.2mg ~（-1））的常规培养基<-1>中增殖活跃，形成由8 ~ 16个小圆柱形细胞串联而成的简单细胞团，细胞内的质体在整个培养期间保持不分化。相比之下，在含有细胞分裂素（BA，1 mg-1 ^<-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造粉体。除了这些特征（无活性的细胞增殖、细胞增大、淀粉体发育）之外，我们发现BY-2细胞在生长素耗尽的培养基中的行为类似于根冠细胞的行为，因为（i）它们容易彼此剥离，（ii）表现出短的寿命，和（iii）显示出更高的分泌活性。结果表明，BY-2细胞特异性分泌4种蛋白，其中1种（SPCT 44）为分泌型过氧化物酶。另外三种蛋白由于N端封闭，无法确定N端肽段序列。通过测定内部肽序列鉴定它们的工作正在进行中。为了分析正常BY-2细胞向根冠样细胞分化过程中基因表达的变化，我们进行了ddRT-PCR和微阵列分析（与RIKEN合作），使用从在常规培养基或改良培养基中培养12小时的BY-2细胞中纯化的mRNA，并鉴定了根据根冠样细胞的分化激活表达的几个基因。结果由RIKEN在BY-2 EST数据库中打开（http：//mrg.psc.riken.go.jp/strc/）。我想利用本研究项目获得的分子标记，进一步分析在改良培养基中根冠细胞和根冠细胞样BY-2细胞之间的相似性。