羽衣甘蓝自交不亲和与自交亲和系种子萌发期DNA甲基化的动态变化

摘要

自交不亲和系植株的种子往往出现退化现象,为了研究种子的退化现象是否与甲基化相关,因此本文采用甲基化敏感扩增多态性(methylation sensitive amplification polymorphism, MSAP)技术,以羽衣甘蓝自交不亲和系9#种子、自交亲和系14#种子为研究对象,对其生长发育过程种子基因组DNA甲基化水平变化情况进行研究。采用改良的 CTAB 法提取种子萌发不同时期 DNA,然后通过 MSAP 分析、统计扩增条带,比较二者之间的差异。对9#种子DNA甲基化状态分析表明,萌发前期(0~2 d)发生甲基化位点数目持续增多,但萌发后期(2~8 d)发生去甲基化的数目大量增加,整个萌发期去甲基化位点数目是甲基化位点数目的11倍,说明9#种子萌发过程中 DNA 甲基化修饰是基因表达的重要调控方式之一；在相同发育时期,9#在总甲基化、全甲基化、半甲基化水平上均不同程度高于14#。随着种子的萌发,9#全甲基化水平明显上升,半甲基化水平几乎不变,而14#变化趋势与9#相反,半甲基化水平明显上升,全甲基化水平几乎不变。%The seed of self-incompatible line often degraded. This study aims to clarify the relationship between the degradation of the seeds and methylation. In this experiment, methylation sensitive amplification polymorphism (MSAP) was used to study the status and patterns of the DNA methylation at different periods in seed germination of self-incompatible line 9# and self-compatible line 14#. The improved CTAB method is adopted to extract seed germination in different periods of DNA, and then through the MSAP analysis, statistical amplification bands, compare the differences between 9# and 14#. The results are shown as follows:DNA methylation modification throughout the whole process of seed germination of 9#, at the early stage of the germination (0 to 2 days) methylation sites continued to increase;at the later stage (2 to 8 days) demethylation increased appar-ently, and eventually the number of demethylation was 11 times more than methylation. It was proved that DNA methylation modification was an important way to regulate the gene expression during seed germination of self-incompatible line 9#. Self-incompatible line 9# and self-compatible line 14# had different DNA methylation status clearly at the respectively periods of 0 and 2 days after seed germination. The proportion of total methylation, full-methylation and semi-methylation of 9# was all higher than of 14# at the same period. As seedings continued to grow after germination, in 9#, the proportion of full-methylation increased clearly and almost that of semi-methylation did not change, while in 14#, the proportion of semi-methylation increased clearly and almost that of full-methylation did not change.