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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。
The DNA methylation of Euphorbia humilis seedlings, embryogenic callus and regeneration seedling was studied by methylation-sensitive amplified polymorphism (MSAP). Ninety-four pairs of primers were screened from 64 pairs of primers, and a total of 506 bands were amplified. The results showed that the methylation levels of genome-wide DNA of seedlings, embryogenic callus and regenerated seedlings were 31.42% and 27.86% And 29.05% respectively. There were 175 bands with methylation variation among the three samples, with a mutation rate of 34.58%. Somatic embryos induced the formation of embryogenic callus in the process, the methylation level decreased, and in the regeneration of seedlings have been restored, and the field close to. At the outer cytosine methylation level, the methylation level of embryogenic callus increased, and could be partially maintained in the regenerated shoots. In addition, of the 175 bands, there were 62 DNA methylation patterns of regenerated seedlings, accounting for 35.43% of the total mutated bands, while the same DNA methylation pattern as that of embryogenic callus 59, accounting for 33.71%. Partial methylated variant bands were recovered, resulting in eight valid genomic DNA sequences. BLASTn alignment analysis showed that DNA methylation modification existed in many DNA sequences including the resistance gene, protein kinase and plastid gene in the heather genome.