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以棉花杂交种中棉所29为材料,用甲基化敏感扩增多态性(methylation sensitive amplification polymorphism,MSAP)分析法结合毛细管电泳检测技术进行甲基化鉴定,以初步探讨棉花耐盐的分子机理.应用24个引物组合,中棉所29在0.4%盐水胁迫及清水对照下,平均每引物组合检测甲基化位点数分别为69.2和56.7,差异达显著水平.盐胁迫下的DNA甲基化水平与清水对照下相比,52.6%位点表现出甲基化水平提高,即发生了超甲基化;19.7%位点甲基化水平降低,即表现为次甲基化;二者差异达极显著水平.研究结果表明,中棉所29盐胁迫后发生了广泛的DNA甲基化变化,包括超甲基化和次甲基化,以及其它甲基化类型的转变;发生超甲基化位点极显著地多于发生次甲基化位点.盐胁迫下的中棉所29与对照相比,DNA总体甲基化水平显著提高,暗示中棉所29有提高基因组甲基化水平以应对盐胁迫的潜在机制,棉花基因组整体甲基化水平的提高可能与棉花对盐胁迫的耐受性起重要作用.本研究中,甲基化序列的初步克隆及比对分析表明,盐胁迫前后多个ATP合成相关基因甲基化程度维持在同一水平,其表达不受甲基化影响,这也可能是中棉所29耐盐性较强,在一定时间盐处理后能维持正常生长的原因之一.
A cotton hybrid, Zhongmiansuo 29, was used for methylation identification by methylation-sensitive amplification polymorphism (MSAP) analysis combined with capillary electrophoresis. Mechanism.Using 24 primer combinations, the average number of methylated sites detected by primer combinations was 69.2 and 56.7, respectively, under the condition of 0.4% saline stress and water control, and the difference was significant at the level of DNA methyl The level of methylation was increased in 52.6% of the cases compared with that in the control of water, that is, hypermethylation occurred; the methylation level decreased in 19.7% of the cases, that is, methylated; Reached the extremely significant level.The results showed that there was a wide range of DNA methylation changes, including hypermethylation and methylation, and other types of methylation, Compared with the control, the overall methylation level of DNA was significantly increased, suggesting that CIMC 29 had increased methylation level of genomic DNA To cope with the underlying mechanism of salt stress, cotton-based The improvement of overall methylation level may play an important role in the tolerance to salt stress.In this study, the preliminary cloning and alignment analysis of methylation sequences showed that the number of ATP-related genes such as methyl The degree of chemical remains at the same level, and its expression is not affected by methylation. This may also be one of the reasons for the strong salt tolerance of CIMC 29 and the normal growth after salt treatment for a certain period of time.