Miklashevichs E, Czaja I, Cordeiro A, Prinsen E, Schell J, Walden R
Max-Planck-Institut für Züchtungsforschung, Köln, Germany.
Plant J. 1997 Sep;12(3):489-98. doi: 10.1046/j.1365-313x.1997.00489.x.
Activation T-DNA tagging was used to generate four cytokinin-independent (cyi1-4) tobacco cell lines. Plants regenerated from the mutant lines displayed similar phenotypes: reduced apical dominance, poorly developed roots, delayed growth and flowering, and male and female sterility. Tissue culture experiments demonstrated that the mutations in the different lines uncouple cell proliferation from the effects of both cytokinin and auxin. No significant increase of cytokinin or auxin was found in transgenic calli in comparison with untransformed callus. The functional plant sequence tagged in one of the mutant lines, cyi1, was used to isolate an active cDNA, cyi1a, that was able to trigger cytokinin- and auxin-independent protoplast division. Northern analysis shows that the transcript corresponding to cyi1a accumulates to high levels in the untransformed protoplasts shortly before the onset of cell division, and that these levels decrease when protoplasts reach maximum rates of cell division. A small putative open reading frame, starting with the first ATG in cyi1a and encoding a 22 amino acid peptide, has the same activity in tobacco protoplasts as the whole cDNA. This activity is destroyed by a frame shift mutation. Apparently cyi1a encodes a peptide which participates in the events downstream of a joint point of cytokinin and auxin action leading to cell division.
利用激活标签法获得了4个不依赖细胞分裂素的烟草细胞系（cyi1 - 4）。从这些突变系再生的植株表现出相似的表型：顶端优势减弱、根系发育不良、生长和开花延迟以及雄性和雌性不育。组织培养实验表明，不同系中的突变使细胞增殖与细胞分裂素和生长素的作用解偶联。与未转化的愈伤组织相比，转基因愈伤组织中未发现细胞分裂素或生长素的显著增加。在其中一个突变系cyi1中标记的功能植物序列用于分离一个活性cDNA，cyi1a，它能够引发不依赖细胞分裂素和生长素的原生质体分裂。Northern分析表明，与cyi1a对应的转录本在细胞分裂开始前不久在未转化的原生质体中积累到高水平，而当原生质体达到最大细胞分裂速率时这些水平下降。一个小的假定开放阅读框，从cyi1a中的第一个ATG开始，编码一个22个氨基酸的肽，在烟草原生质体中具有与整个cDNA相同的活性。这种活性被移码突变破坏。显然，cyi1a编码一种肽，它参与细胞分裂素和生长素作用导致细胞分裂的交汇点下游的事件。
