Characterization of genes involved in regulation of differentiation using mutant plant cell cultured lines by insertion of transposable elements

在胡萝卜悬浮细胞苯丙氨酸解氨酶基因启动子区发现了Tdc 1的En/Spm转座元件。Tdc 1只有一个长的开放阅读框对应于一个转座酶，但另一个转座酶，这是必要的切除和插入的Tdc 1，在Tdc 1部分编码，这表明Tdc 1应该是一个非自主转座元件Southern分析表明，超过20个拷贝的Tdc 1-like元件可能在胡萝卜基因组中。PCR扩增Tdc家族的其它元件，并进行核苷酸序列分析。胡萝卜基因组中存在Tdc的三个亚家族Tdc-A、Tdc-B和TdcR。Tdc-A1编码两个与玉米En/Spm转座因子部分相似的转座酶，推测Tdc-A1可能是Tdc家族的自主转座因子。Tdc-A的其他变体显示内部缺失，而核苷酸序列没有变化。这些结果表明，Tdc-A在胡萝卜细胞继代培养过程中发生了积极的转座并伴有缺失，从而导致了培养变异。Tdc-B和Tdc-R是Tdc-A的缺失突变体，其核苷酸序列与Tdc-A相比发生了改变，可能在胡萝卜细胞培养建立前发生了转座。

An En/Spm transposable elements of Tdc1 was found in the promoter region of phenylalanine ammonia-lyase gene in carrot suspension cultured cells. Tdc1 had only one long open reading frame corresponding to one transposase, but the other transposase, which is necessary for excision and insertion of Tdc1, was partially encoded in Tdc1, suggesting that Tdc1 should be a non-autonomous transposable element Southern analysis indicates that more than 20 copies of Tdc1-like elements may be in the carrot genome. The other elements of Tdc family were obtained using PCR and the nucleotide sequences were analyzed. Three sub-family of Tdc, Tdc-A, Tdc-B and TdcR, were found in carrot genome. Tdc-A1 encoded two transposases which were partially similar to maize En/Spm transposable element, suggesting that Tdc-A1 might be the autonomous element of Tdc family. The other Variants of Tdc-A showed the internal deletions without changes in nucleotide sequences. These results suggest that Tdc-A actively transposed accompanied with deletion during subculturing of carrot cells, which results in culture variation. Tdc-B and Tdc-R were deletion variants of Tdc-A.Their nucleotide sequences were changed in comparison with Tdc-A.They might transpose before the establishment of carrot cell culture.