Prakash A Pavan, Kush Anil, Lakshmanan Prakash, Kumar Prakash P
Plant Morphogenesis Laboratory, Department of Biological Sciences, The National University of Singapore, 10 Science Drive 4, Singapore 117543.
J Exp Bot. 2003 May;54(386):1361-71. doi: 10.1093/jxb/erg155.
The DNA methylase inhibitors, 5-azacytidine and 5-aza-2'-deoxycytidine inhibited adventitious shoot induction in Petunia leaf cultures. Cytosine methylation status at CCGG sites in shoot- and callus-inducing culture treatments was analysed by coupled restriction enzyme digestion (HpaII or MspI) and random amplification. Two differentially methylated genomic DNA bands from the PCR products were cloned (OPU9-1 and OPU9-2) and sequenced. The open reading frames contained in OPU9-1 and OPU9-2 showed similarity to CDC48 and MADS-box genes, respectively. Cytosine methylation was restored at CCGG sites when the leaf explants were transferred from medium containing the drugs to medium without the drugs, simultaneously recovering the ability to develop adventitious shoot buds. Furthermore, combined bisulphite treatment and restriction analysis revealed differential methylation of CGCG sites in the drug-treated and control cultures. These results demonstrate that cytosine methylation at CCGG and CGCG sites within a MADS-box gene and a CDC48 homologue, among others, shows strong positive correlation with adventitious shoot bud induction in Petunia leaf explants.
DNA甲基化酶抑制剂5-氮杂胞苷和5-氮杂-2'-脱氧胞苷抑制了矮牵牛叶片培养物中不定芽的诱导。通过限制性内切酶联合消化（HpaII或MspI）和随机扩增分析了不定芽诱导培养处理和愈伤组织诱导培养处理中CCGG位点的胞嘧啶甲基化状态。从PCR产物中克隆了两条差异甲基化的基因组DNA条带（OPU9-1和OPU9-2）并进行测序。OPU9-1和OPU9-2中包含的开放阅读框分别与CDC48和MADS-box基因相似。当叶片外植体从含药培养基转移到不含药的培养基时，CCGG位点的胞嘧啶甲基化得以恢复，同时恢复了发育不定芽的能力。此外，亚硫酸氢盐联合处理和限制性分析揭示了药物处理培养物和对照培养物中CGCG位点的差异甲基化。这些结果表明，除其他外，MADS-box基因和CDC48同源物内CCGG和CGCG位点的胞嘧啶甲基化与矮牵牛叶片外植体中不定芽诱导呈强正相关。
