Regulation of plant expression by DNA methylation

5-氮胞苷是一种有效的DNA甲基化抑制剂，在体内暴露于正常的高秆水稻幼苗，可以诱导基因组DNA和矮秆植株的去甲基化。通过DNA文库的差异筛选鉴定了受治疗影响的基因，随后编码了一个小的GTP结合蛋白rgp1的基因。该基因编码226个氨基酸(24.8 kDa)，与裂殖酵母ras相关的ypt3蛋白具有最高的序列相似性。在转化的大肠杆菌中表达的rgp1蛋白rgp1-p25具有明显的GTP结合活性。为了确定rgp1-p25的生理功能，将rgp1的编码区分别从正义和反义两个方向导入烟草。在两个方向上都含有rgp1的转化子表现出明显的表型变化，顶端显性降低，矮化和花发育异常。在反义转化子中，rgp1的烟草同源基因tgp1的表达显著降低，表明其表型变化可能是通过干扰tgp1的mRNA来实现的。在正义转化子中，这些变化可能是由于rgp1-p25的过度生产而引起的。我们推测，分蘖增加可能与植物激素代谢异常或反应途径有关。初步实验表明，在高等植物中，玉米是一种活性细胞分裂素，其含量在转化体和对照之间有显著差异。此外，还发现转化子对外用细胞分裂素的反应不同。这些观察表明，rgp1-p25可能参与了细胞分裂素生物合成的调节，或者更有可能参与了细胞分裂素的细胞内运输。

Exposure of normal, tall rice (Oryza sativa) seedlings to 5-azacytidine, a powerful inhibitor of DNA methylation in vivo, induced both demethylation of genomic DNA and dwarf plants. Genes that had been affected by treatment were identified by differential screening of a DNA library, and a gene encoding a small GTP-binding protein, rgp1, was subsequently. The cDNA or rgp1 was found to encode a deduced protein of 226 amino acids (24.8kDa) with highest sequence similarity to the ras-related ypt3 protein of fission yeast. The rgp1 protein, rgp1-p25, expressed in transformed E.coli, clearly showed GTP-binding activity. To determine the physiological role of rgp1-p25, the coding region of rgp1 was introduced into tobacco plants in both sense and anti-sense orientations. Transformants, with rgp1 in either orientation, showed distinct phenotypic changes; reduced apical dominance, dwarfism and abnormal flower development. The expression of tgp1, a tobacco homologue of rgp1, was markedly reduced in anti-sense transformants, indicating that tha phenotypic changes may have been mediated by interference with tgp1, mRNA. In sense transformants, the changes may have been induced by over-production of rgp1-p25. We speculated that the increased tillering may be related to abnormal phytohormone metabolism or response pathways. A preliminaly experiment clearly showed that the content of zeation, one of the active cytokinins in higher plants, differed significantly between transformants and controls. Furthermore, transformants were found to respond differentially to externally-applied cytokinins. These observations indicate that the rgp1-p25 may be involved in either the regulation of cytokinin biosynthesis or, more probably, in its intracellular transport.