不同感温性甘蓝型冬油菜DNA甲基化差异及At4g02000-like结构预测

为研究甘蓝型冬油菜(Brassica napus L.)在低温胁迫后DNA甲基化水平及模式的变化情况,以强抗寒的15TS306和14NS52-3等13个甘蓝型冬油菜为材料,利用甲基化敏感扩增多态性技术,选用12对引物组合对低温(4℃)胁迫后的冬油菜DNA甲基化水平及模式的变化情况进行检测,并对差异片段进行序列比对及克隆。结果发现:低温胁迫后,弱抗寒的14美切实7、14美切实16、14美切实20、14美切实3和美切实38等品系DNA甲基化水平有所升高,且具有较高的甲基化程度;而强抗寒的15TS306、14NS52-3、15TS309、14NS54-7和15TS312等品系去甲基化程度较高。经过对22条DNA甲基化特异片段的序列分析,有16条片段序列与已知和假定功能的酶及蛋白具有同源性,其中以At4g02000-like蛋白变化最为明显。克隆及生物信息学分析显示,At4g02000-like蛋白等电点9. 20,相对分子质量38. 89kD。甘蓝型冬油菜受低温胁迫后,抗寒性强的品种DNA甲基化水平降低,以去甲基化为主;抗寒性弱的材料DNA甲基化水平升高;而且油菜对低温环境的适应性与某些特定基因不同的甲基化模式密切相关。

Difference of DNA methylation in winter rapeseed (Brassica napus L.) with temperature sensitivity and prediction of At4g02000-like protein

 To better understand the cold-resistance of winter rapeseed (Brassica napus L.), DNA methylation were investigated using MSAP (methylation sensitive amplified polymorphism) technology on 13 materials with different cold resistance under low temperature (4oC) stress. MSAP results showed that, after 4oC treatment, the weak cold-resistance lines (14 Meiqieshi 7, 14 Meiqieshi 16, 14 Meiqieshi 20, 14 Meiqieshi 3 and Meiqieshi 38 had increased DNA methylation level and had higher methylation degree. The strong cold-resistant lines (15TS306, 14NS52-3, 15TS309, 14NS54-7 and 15TS312) had higher degree of demethylation. Sequence analysis of the differentially methylated fragments showed that 16 of 22 fragments were similar to the known and putative functional enzymes. Among them, At4g02000-like protein had the greatest variation. Cloning and bioinformatics results showed that its isoelectric point was 9.49 and the relative molecular weight was 39.2525kD. In sumarry, after low temperature stress, the strong cold-resistant lines had decreased DNA methylation level with dominant demethylation, while the weak cold-resistance lines had increased DNA methylation level with dominant methylation. In these winter rapeseeds, cold adaptability showed close correlation between differentially methylation patterns of some specific genes.