中国水产科学2016，Vol.23Issue(6)：1227-1235,9.DOI:10.3724/SP.J.1118.2016.16077
马氏珠母贝外套膜不同区域基因组DNA甲基化MSAP分析
Analysis of genomic DNA methylation on different regions of mantle tissue fromPinctada martensii by methylation-sensitive amplification polymorphism
摘要
Abstract
DNA methylation is closely linked to biological events, including chromatin inactivation, transgene si-lencing, genomic imprinting and control of parasitic DNA elements. Because of its efficiency and competence, the methylation-sensitive amplification polymorphism (MSAP) technique has been used increasingly in genomic DNA and individual functional gene studies to analyze DNA methylation levels. In this research, MSAP technology was used to analyze the methylation levels of mantle tissues fromPinctada martensii, including the mantle edge (Me), mantle pallium (Mp) and mantle central (Mc). Recycling the methylation of specific fragments to be sequenced, comparative analysis and selection the target gene, then used the Real time PCR to analyze the target gene. Results showed that (1163.25±124.34) DNA bands were clear and repetitive by using 15 pairs of primers. Among them, Me had (401.00±40.37) bands, Mp had (380.63±52.39) bands and Mc had (381.63±53.57) bands, and there was no significant difference (P>0.05). The percentages of methylation levels were (17.07±2.19)% in Me, (15.48±2.34)% in Mp and (19.61±2.88)% in Mc (P<0.05). The methylation levels from high to low were Mc>Me>Mp. Methyla-tion patterns included fully methylated sites, hemi-methylated sites and non-methylated sites. The experiment re-sults showed that the percentages of fully methylated sites were higher than hemi-methylated sites in all areas of the mantle, indicating that the methylation pattern in theP.martensii genome was mainly a CpG island. After recovering and sequencing the specific bands, we found eight gene sequences, which were methylated by Blast. Of these, there were three sequences with homologous sequences by Local Blast with genome, which were 40S ribosomal protein SA, interference hedgehog and Zinc finger protein castor by gene annotations. Interference hedgehog (iHog) was the target gene; real-time PCR showed thatiHog was expressed in Me, Mp and Mc, with the highest expression level in Me and the lowest expression level in Mc (P<0.05). This indicates that the expression ofiHog in Mc was inhibited by DNA methylation. These results confirm that the mantle tissues Me, Mp and Mc have dif-ferent methylation levels. DNA methylation could also play a role in the regulation of gene expression. The tech-nique helps us to understand the relationship between methylation and expression regulation, and also provides a theoretical basis to elaborate the mechanisms underlying biomineralization and immune response inP.martensii.关键词
马氏珠母贝/外套膜/MSAP技术/DNA甲基化/表达调控Key words
Pinctada martensii/mantle/methylation-sensitive amplification polymorphism (MSAP)/DNA me-thylation/expression regulation分类
农业科技引用本文复制引用
罗少杰,邓岳文,郑哲,焦钰,王庆恒..马氏珠母贝外套膜不同区域基因组DNA甲基化MSAP分析[J].中国水产科学,2016,23(6):1227-1235,9.基金项目
国家自然科学基金资助项目(41206141,31372526) （41206141,31372526）
广东省科技计划项目(2013B020201002) （2013B020201002）
