| 马氏珠母贝外套膜不同区域基因组DNA甲基化MSAP分析 |
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| 引用本文： | 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. |
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| 作者姓名： | 罗少杰 邓岳文 郑哲 焦钰 王庆恒 |
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| 作者单位： | 1. 广东海洋大学水产学院,广东湛江,524025;2. 广东海洋大学水产学院，广东湛江 524025; 广东省珍珠养殖与加工工程技术研究中心，广东湛江 524025 |
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| 基金项目： | 国家自然科学基金资助项目(41206141;31372526)，广东省科技计划项目(2013B020201002) |
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| 摘 要： | 通过甲基化敏感多态性扩增(methylation-sensitive amplification polymorphism,MSAP)技术,检测马氏珠母贝(Pinctada martensii)外套膜的边缘膜区(mantle edge,Me)、套膜区(mantle pallial,Mp)和中央膜区(mantle central,Mc)的基因组DNA甲基化修饰水平;回收具特异性的清晰甲基化修饰片段进行测序、比对分析并筛选基因;利用荧光定量PCR对筛选基因进行定量分析。结果显示:(1)利用15对引物进行扩增实验,平均每个个体外套膜3个区域能够产生(1163.25±124.34)条清晰可辨的条带,其中Me、Mp和Mc分别得到(401.00±40.37)条、(380.63±52.39)条和(381.63±53.57)条扩增条带,各组织甲基化总条数差异不显著(P0.05);Me、Mp和Mc的基因组甲基化水平分别为(17.07±2.19)%、(15.48±2.34)%和(19.61±2.88)%,Mc和Mp具有显著性差异(P0.05);组织间的基因组甲基化水平由高到低排列依次是McMeMp。(2)对特异性片段进行回收、测序后,经在线及本地Blast比对,得到8条存在甲基化修饰的基因序列,其中3条有同源序列,基因注释为40S核糖体蛋白SA(40S ribosomal protein SA)、iHog(interference hedgehog)、锌指蛋白(zinc finger protein castor)。iHog仅在中央膜上具有全甲基化修饰,且E值较低,为筛选的目的基因。(3)荧光定量结果表明iHog在Me、Mp和Mc中均有表达,以Me表达量最高,Mc表达量最低,差异显著(P0.05)。我们推测iHog的DNA序列的甲基化修饰抑制了该基因在Mc组织中的表达。综上研究结果表明,马氏珠母贝外套膜3个区域的甲基化修饰水平不一,且DNA甲基化在基因表达调控中具有作用,这对深入研究珍珠贝生物矿化和免疫反应机制具有一定的参考意义。
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| 关 键 词： | 马氏珠母贝 外套膜 MSAP技术 DNA甲基化 表达调控 |
| 修稿时间： | 2016/11/9 0:00:00 |
Analysis of genomic DNA methylation on different regions of mantle tissue from Pinctada martensii by methylation-sensitive amplification polymorphism |
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| LUO Shaojie,DENG Yuewen,ZHENG Zhe,JIAO Yu,WANG Qingheng.Analysis of genomic DNA methylation on different regions of mantle tissue from Pinctada martensii by methylation-sensitive amplification polymorphism[J].Journal of Fishery Sciences of China,2016,23(6):1227-1235. |
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| Authors: | LUO Shaojie DENG Yuewen ZHENG Zhe JIAO Yu WANG Qingheng |
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| Institution: | 1. Fisheries College, Guangdong Ocean University, Zhanjiang 524025, China;2. Guangdong Technology Research Center for Pearl Aquaculture and Process, Zhanjiang 524025, China |
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| 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. |
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| Keywords: | Pinctada martensii mantle methylation-sensitive amplification polymorphism (MSAP) DNA me-thylation expression regulation |
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