背景:DNA甲基化可以迅速变化以调节应激反应基因的表达。以往的研究表明,冬油菜在不同的选择环境下驯化后,其抗寒性存在显著差异;然而,对其抗寒性形成的表观遗传调控机制知之甚少。
方法:四种冬季油菜籽材料(\'CT-2360\',\'MXW-1\',\'2018-FJT\',选择在不同环境下驯化的\'DT-7\'),使用甲基化敏感的扩增多态性技术,用60对引物对分析低温下的DNA甲基化水平和模式变化。
结果:共筛选出18对多态性良好的引物,扩增了1426条清晰的条带,有594个甲基化位点,占扩增条带总数的41.65%。低温处理后四种材料的总甲基化率降低,其中“CT-2360”的DNA甲基化水平高于其他三种材料;甲基化模式分析显示,去甲基化程度高于“MXW-1”中的甲基化程度,\'2018-FJT\',和\'DT-7\',是22.99%,19.77%,和24.35%,分别,CT-2360中的甲基化事件占22.95%,占主导地位。随机抽取53个多态性甲基化DNA片段,29个克隆片段与已知功能的基因同源。候选基因VQ22和LOC103871127验证了低温处理前后不同表达模式的存在。
结论:我们的工作暗示了DNA甲基化在冬油菜抗寒性形成中的关键作用。这些结果为油菜对低温的适应表观遗传调控机制提供了全面的见解。鉴定出的差异甲基化基因也可作为耐冬品种多边育种的重要遗传资源。
BACKGROUND: DNA methylation can change rapidly to regulate the expression of stress-responsive genes. Previous studies have shown that there are significant differences in the cold resistance of winter rapeseed (Brassica rapa L.) after being domesticated in different selection environments; however, little is known about the epigenetic regulatory mechanisms of its cold resistance formation.
METHODS: Four winter rapeseed materials (\'CT-2360\', \'MXW-1\', \'2018-FJT\', and \'DT-7\') domesticated in different environments were selected to analyze the DNA methylation level and pattern changes under low temperature using methylation-sensitive amplified polymorphism technology with 60 primer pairs.
RESULTS: A total of 18 pairs of primers with good polymorphism were screened, and 1426 clear bands were amplified, with 594 methylation sites, accounting for 41.65% of the total amplified bands. The total methylation ratios of the four materials were reduced after low-temperature treatment, in which the DNA methylation level of \'CT-2360\' was higher than that of the other three materials; the analysis of methylation patterns revealed that the degree of demethylation was higher than that of methylation in \'MXW-1\', \'2018-FJT\', and \'DT-7\', which were 22.99%, 19.77%, and 24.35%, respectively, and that the methylation events in \'CT-2360\' were predominantly dominant at 22.95%. Fifty-three polymorphic methylated DNA fragments were randomly selected and further analyzed, and twenty-nine of the cloned fragments were homologous to genes with known functions. The candidate genes VQ22 and LOC103871127 verified the existence of different expressive patterns before and after low-temperature treatment.
CONCLUSIONS: Our work implies the critical role of DNA methylation in the formation of cold resistance in winter rapeseed. These results provide a comprehensive insight into the adaptation epigenetic regulatory mechanism of Brassica rapa L. to low temperature, and the identified differentially methylated genes can also be used as important genetic resources for the multilateral breeding of winter-resistant varieties.