野生和“黄海1号”中国明对虾不同组织基因组DNA的MSAP分析

MSAP analysis of genomic DNA in the tissues of wild and "Huanghai No.1" Fenneropenaeus chinensis

为了从表观遗传学角度讨论中国明对虾(Fenneropenaeus chinensis)野生群体和人工选育新品种"黄海1号"不同组织间甲基化水平和多态性差异,应用甲基化敏感扩增多态性(mehylation sensitive amplified polymorphism,MSAP)分别对野生群体组中国明对虾和"黄海1号"肌肉、鳃、血液3种组织样品基因组DNA的CCGG甲基化水平进行对比分析,试图从表观遗传学角度探讨影响中国明对虾生长性状的分子机制。采用30对引物进行选择性扩增,经聚丙烯酰胺凝胶电泳(Polyacrylamide Gel Electrophoresis,PAGE)带型结果显示,野生群体组中国明对虾肌肉、鳃和血液的甲基化比例分别为23.1%,22.3%和19.7%;而"黄海1号"肌肉、鳃和血液的甲基化比例分别为21.4%、19.6%和18.9%。野生群体组中国明对虾和"黄海1号"同一组织间的甲基化水平和甲基化多态性水平不同,肌肉、鳃和血液不同组织间的甲基化水平和甲基化多态性水平亦不同。DNA甲基化多态性带型分析显示,鳃组织的甲基化水平和多态性水平在野生群体组中国明对虾和"黄海1号"间变化趋势最大,肌肉最稳定。本研究旨为甲基化修饰与中国明对虾生长性状间的相关性研究提供依据。

We evaluated the epigenetic molecular mechanisms controlling the growth of Fenneropenaeus chinensis We used MSAP （Methylation-Sensitive Amplification Polymorphism） to analyze methylation patterns of genomic DNA in the muscle, gill, and blood of wild and cultivated Echinensis ＂Huanghai No. 1＂, a strain selected for faster growth rates and improved disease resistance. DNA methylation is closely linked to biological events, including chromatin inactivation, transgene silencing, genomic imprinting, and control of parasitic DNA elements. Because of its efficiency and competence, the MSAP technique has been increasingly used in genomic DNA or individual functional genes studies to analyze DNA methylation levels. We used 30 pairs of selective-amplification primers. Using polyacrylamide gel electrophoresis, we estimated the methylation ratios in the muscle, gill, and blood of wild stock were 23.1%, 22.3%, and 19.7%, respectively, and 21.4%, 19.6%, and 18.9%, respectively, in cultured shrimps. These levels are much lower than in some plants and animals, perhapsbecause of differences in the amount of genomic DNA. The methylation levels at the CCGG site differed among different tissues of the same stock and between the same tissue among different stocks. The level of methylation in genomic DNA was always higher in the organs than in the blood. We observed variation in the DNA polymorphic methylation between wild and ＂Huanghai No. 1＂ stocks in the blood and gill, but not in the muscle. Furthermore, polymorphic methylation was associated with demethylation and methylation of CCGG loci. Additional research is needed to understand the biological meaning of variation in methylation among CCGG loci. Our approach provides a basis for further stud- ies on the mechanism of methylation-mediated DNA methyltransferases in F. chinensis.