盐胁迫下棉花基因组基于毛细管电泳的MSAP分析

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．

Analysis of Methylation-sensitive Amplified Polymorphism in Cotton Genome under Salt Stress Based on Capillary Electrophoresis

In this research, methylation-sensitive amplification polymorphism (MSAP) based on capillary electrophoresis was used to analyze DNA methylation level in a cotton hybrid CCRI 29. A total 24 primer combinations were used, the methylated CCGG loci per primer combination were detected respectively as 69.2 and 56.7 in CCRI 29 treated with 0.4% of salt water and control. A 52.6% of the loci showed increased methylation level and 19.7% decreased under salt stress, indicating that both hypermethylation and hypomethylation occurred| but hypermethylation was more significant. The results showed that salt stress induced DNA methylation changes in CCRI 29. Increased DNA methylation under salt stress could be a potential mechanism for the cotton genome to respond to salt stress. Subsequent cloning and analysis of methylated DNA sequences showed that several ATP synthase subunit genes maintained a stable methylation level and not changed following salt stress. This partial explained that CCRI 29 was salt tolerant and maintained normal growth in salt stress for a certain period.