Characterization of a Cytokinin-Repressed Gene CR20

为了了解细胞分裂素作用的分子机制，我们试图确定CR20基因的结构和性质，该基因在黄瓜黄化子叶中被迅速抑制。对CR20基因的克隆和基因组分析表明，该基因由三个外显子组成，至少有三种类型的转录本是通过第二个内含子的选择性剪接而产生的。然而，这些记录中没有一个包括一个长的开放阅读框架。TESTCODE分析将编码蛋白质的DNA与非编码DNA区分开来，结果预测CR20 RNA将是一个非编码序列。以CR20为探针从拟南芥的cDNA文库中分离得到的一个AtCR20-1也被TESTCODE预测为非编码序列，在CR20和AtCR20-1的RNA中有一个180nt的区域被很好地保守，似乎形成了稳定的茎。这些结果表明，CR20和AtCR20-1的转录产物可能是非编码的功能RNA。为了研究CR20的功能，将含有第二内含子的CR20A基因与不含内含子的CR20-C基因融合到CaMV 35S启动子上，并导入烟草。其中一个CR-20-A转化子表现出沿中脉的黄化和顶端分生组织活性的丧失。另一种植物的叶子比普通植物的绿叶浅。然而，CR20-C转化子与普通植株相比没有表现出不同的特征。由于CR20基因在绿化过程中被抑制，这是已知的细胞分裂素刺激的，转化CR20-A引起的黄化将是显著的。目前正在研究这些变化与CR20基因表达之间的相关性。

As an approach to the understanding of the molecular mechanism of the action of cytokinins, an attempt was made to determine the structure and properties of the CR20 gene, which is rapidly repressed after application of this hormone to etiolated cotyledons of cucumber. Analysis of cDNA and genomic clones of CR20 revealed that this gene consisted of three exons At least three types of transcript were generated by alternative splicing of the second intron. However, none of these transcripts included a long open reading frame. Results of the TESTCODE analysis, which distinguishes protein-coding DNA from noncoding DNA,predicted that CR20 RNA would be a noncoding sequence. A cDNA AtCR20-1, which was isolated from a cDNA library of Arabidopsis thaliana with the CR20 cDNA as a probe, was also predicted to be a noncoding sequence by the TESTCODE.A region of 180 nt was well conserved in CR20 and AtCR20-1 RNAs, and seems to form stable stems. These suggest that the transcripts of CR20 and AtCR20-1 may be noncoding functional RNAs. In order to study the function of CR20, CR20A cDNA which contains the second intron and CR20-C cDNA which does not contain the intron were fused to a CaMV 35S promoter, and introduced into tobacco plants. One of the CR-20-A transformants showed chlorosis along midribs and loss of the activity of the apical meristem. Another one had lighter green leaves than normal plants. However, CR20-C transformants showed no different features compared with normal plants. Chlorosis induced by transformation with CR20-A would be significant, as the CR20 gene was repressed during greening which is known to be stimulated by cytokinins. Correlation between these changes and the expression of the CR20 gene is now being examined.