《植物生理学报》 2013, 49(12): 1413-1420

欧石楠体细胞胚发生过程中的DNA甲基化

姚培娟, 李际红*, 亓晓, 邢世岩

山东农业大学林学院, 农业生态与环境重点实验室, 山东泰安271018

通信作者：李际红；E-mail: jhli4856@163.com；Tel: 1861522637

摘　要：

利用甲基化敏感扩增多态性(MSAP)方法, 对欧石楠大田苗、胚性愈伤组织和再生苗的DNA甲基化进行了研究。从64对选扩增引物中筛选出19对, 共扩增得到506条带, 统计显示, 大田苗、胚性愈伤组织和再生苗的全基因组DNA甲基化水平分别为31.42%、27.86%和29.05%, 3种试材发生甲基化变异的有175条带, 变异率为34.58%。体细胞胚诱导形成胚性愈伤组织过程中, 甲基化水平降低, 而在再生苗中有所恢复, 与大田苗接近。在外侧胞嘧啶甲基化水平上, 胚性愈伤组织的甲基化水平有所增加, 且在再生苗中可部分维持。另外, 在175条变异带中, 再生苗恢复到大田苗DNA甲基化模式的有62条, 占总变异条带的35.43%, 而与胚性愈伤组织维持相同DNA甲基化模式的有59条, 占33.71%。回收部分甲基化变异条带, 最终得到8条有效的基因组DNA序列。BLASTn比对分析表明, 在欧石楠基因组中, 包括抗性基因、蛋白激酶、质体基因等在内的多种DNA序列均存在DNA甲基化修饰现象。

关键词：欧石楠; DNA甲基化; 体细胞胚发生; 胚性愈伤组织; 再生苗

收稿：2013-10-17   修定：2013-11-12

资助：山东省泰安市科技局项目(2011-2013)、山东省东营市科技局项目(2009-2011)、山东省泰安市大学生科技引领创新计划项目(2008D2010及2009D1009)

DNA Methylation in Somatic Embryogenesis of Erica carnea L.

YAO Pei-Juan, LI Ji-Hong*, QI Xiao, XING Shi-Yan

Key Laboratory of Agricultural Ecology and Environment, College of Forestry, Shandong Agricultural University, Tai’an, Shandong 271018, China

Corresponding author: LI Ji-Hong; E-mail: jhli4856@163.com; Tel: 1861522637

Abstract:

The extent of DNA methylation polymorphisms were evaluated in Erica carnea field seedlings, embryogenic calli and regenerated seedlings using methylation-sensitive amplified polymorphism (MSAP) technique. In all, 506 bands were produced by 19 selective primer pairs. The percentages of whole-genomic DNA methylation in field seedlings, embryogenic calli and regenerated seedlings were 31.42%, 27.86% and 29.05%, respectively. There were 175 methylation alteration bands (34.58%) among three kinds of materials. It was found that methylation level of embryogenic calli reduced in the process of somatic embryo induction and recovered in the regenerated seedlings, whose methylation level was close to field seedlings. The lateral cytosine methylation level of embryogenic calli was increased and could partially maintain in regenerated seedlings. In addition, in the total of 175 alteration bands, 62 bands (35.43%) from regenerated seedlings recovered to DNA methylation patterns of field seedlings, while 59 bands (33.71%) from regenerated seedlings maintained the same DNA methylation patterns of embryogenic calli. Eight methylated DNA fragments were isolated and sequenced by extracting part of the amplified sites. BLASTn analysis indicated that DNA methylated modification existed in multiple types of DNA fragments, including resistance gene sequences, protein kinase, plastogene, etc.

Key words: Erica carnea; DNA methylation; somatic embryogenesis; embryogenic calli; regenerated seedlings