CAJ | 학술논문

为了获得黑麦基因组DNA甲基化修饰水平、模式及位点等表观遗传信息,采用EcoR Ⅰ和Hpa Ⅱ / Msp Ⅰ双酶切建立适合于黑麦基因组的"甲基化敏感扩增多态性"(Methylation sensitive amplification polymorphism,MSAP)分析体系,在全基因组水平检测黑麦DNA甲基化修饰位点.以12对MSAP引物进行选择性扩增,共检测到甲基化修饰位点226个,"CCGG/GGCC"位点甲基化修饰比例为51.72%.对部分黑麦基因组甲基化修饰位点进行回收,最终分离了22条存在甲基化修饰的基因组DNA序列.BLAST比对分析结果表明,黑麦基因组中包括转座子序列、散在重复序列以及单拷贝蛋白质编码序列在内的多种类型DNA序列中均存在DNA甲基化修饰现象.同时发现,在甲基化检出序列中都存在明显的"CpG"二核苷酸成簇富集现象,这些区域分布与MSAP分析结果相一致.在此基础上,对应用MSAP技术分离黑麦基因组DNA甲基化修饰位点的有效性以及黑麦基因组序列中DNA甲基化修饰潜在位点分布特征和生物意义进行了讨论.
위료획득흑맥기인조DNA갑기화수식수평、모식급위점등표관유전신식,채용EcoR Ⅰ화Hpa Ⅱ / Msp Ⅰ쌍매절건립괄합우흑맥기인조적"갑기화민감확증다태성"(Methylation sensitive amplification polymorphism,MSAP)분석체계,재전기인조수평검측흑맥DNA갑기화수식위점.이12대MSAP인물진행선택성확증,공검측도갑기화수식위점226개,"CCGG/GGCC"위점갑기화수식비례위51.72%.대부분흑맥기인조갑기화수식위점진행회수,최종분리료22조존재갑기화수식적기인조DNA서렬.BLAST비대분석결과표명,흑맥기인조중포괄전좌자서렬、산재중복서렬이급단고패단백질편마서렬재내적다충류형DNA서렬중균존재DNA갑기화수식현상.동시발현,재갑기화검출서렬중도존재명현적"CpG"이핵감산성족부집현상,저사구역분포여MSAP분석결과상일치.재차기출상,대응용MSAP기술분리흑맥기인조DNA갑기화수식위점적유효성이급흑맥기인조서렬중DNA갑기화수식잠재위점분포특정화생물의의진행료토론.
In this study, the double digestion of EcoR Ⅰ and Hpa Ⅱ/Msp Ⅰ was used to construct the high-density rye (Secale cereale) genomic methylation-sensitive amplification polymorphism fingerprint and detected the methylated sites at the whole genome level. By using 12 pairs of selective primers, a total of 226 methylated sites was detected, which represent 51.72% ratio of methylation modification at 'CCGG/GGCC' site in rye genome. Some of methylated sites were extracted and sequenced. Further sequence analysis showed the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and dispersed repetitive sequences, and low-copy protein coding sequences. The region of CpG rich cluster with typical CpG island sequence features was also detected in the sequence of rye methylated sites. Based on these experimental evidences, the effectiveness to isolate rye methylated site by MSAP and the distribution features of CpG island like sequence in rye genome were discussed.