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

在胡萝卜悬浮培养细胞苯丙氨酸解氨酶基因的启动子区域发现了Tdc1的EN/SPM转座元件。Tdc1只有一个对应于一个转座酶的长的开放阅读框，但Tdc1中的另一个转座酶是切割和插入Tdc1所必需的，这表明Tdc1应该是一个非自主转座元件。Southern分析表明，胡萝卜基因组中可能存在超过20个拷贝的Tdc1类似元件。用聚合酶链式反应获得TDC家族的其他元件，并对其核苷酸序列进行分析。在胡萝卜基因组中发现了三个TDC亚家族：TDC-A、TDC-B和TDCR。TDC-A1编码两个转座酶，与玉米EN/SPM转座元件部分相似，提示TDC-A1可能是TDC家族的自主元件。TDC-A的其他变异体在核苷酸序列没有改变的情况下显示了内部缺失。这些结果表明，在胡萝卜细胞继代培养过程中，TDC-A的活性转座伴随着缺失，从而导致了培养上的变异。TDC-B和TDC-R是TDC-A的缺失变异体，与TDC-A相比，它们的核苷酸序列发生了变化，在胡萝卜细胞培养建立之前，它们可能转座。

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.