焦钰,梁金婵,谷泽丰,张鹏飞,李榕娇,罗少杰

• 1:广东海洋大学水产学院
• 2:广东省珍珠养殖与加工工程技术研究中心

摘要(Abstract)：

【目的】研究马氏珠母贝(Pinctada fucata martensii)血细胞基因组DNA的甲基化修饰情况及其在脂多糖(LPS)刺激后的变化,为探究甲基化修饰在珍珠贝免疫防御中的作用提供理论基础。【方法】运用甲基化敏感扩增多态性法(MSAP)分析马氏珠母贝血细胞(正常养殖贝)以及注射LPS后(6、12、24 h)基因组CCGG位点甲基化的修饰水平变化,并对甲基化修饰位点进行回收测序。【结果】共获得1 102个清晰可辨的DNA条带,其中全甲基化位点条带215个,半甲基化位点条带127个,非甲基化位点条带760个。对照组血细胞基因组CCGG位点的甲基化修饰比例为23.76%,注射LPS后,甲基化比例为28.10%～48.25%,注射6 h时最低,12 h时最高;对特异性片段测序后,经Blast比对,得到5条存在甲基化修饰的基因序列,其中2条具有注释信息,分别是干扰素调控因子(Interferon regulatory factor)和磷脂酶BL2(Putative phospholipase B-like 2)。【结论】马氏珠母贝血细胞基因组CCGG位点的甲基化修饰参与调控LPS诱导的免疫反应,在免疫防御中发挥重要作用。

关键词(KeyWords)： 马氏珠母贝;DNA甲基化;免疫防御;甲基化敏感扩增多态性法(MSAP)

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31672626);; 大学生创新创业训练项目(CXXL2015041)

作者(Author): 焦钰,梁金婵,谷泽丰,张鹏飞,李榕娇,罗少杰

参考文献(References)：

• [1] ARNAUD-HAOND S, GOYARD E, VONAU V, et al.Pearl formation:persistence of the graft during the entire process of biomineralization[J]. Marine biotechnology(New York), 2007, 9:113-116. doi:/10.1007/s10126-006-6033-5.
• [2] LI J, CHEN J H, ZHANG Y, et al. Expression of allograft inflammatory factor-1(AIF-1)in response to bacterial challenge and tissue injury in the pearl oyster, Pinctada martensii[J]. Fish&Shellfish Immunology, 2013, 34:365-371. doi:/10.1016/j.fsi.2012.11.012.
• [3] CHEN J H, SHU X, DENG Y W, et al. Cloning of a novel glutathione S-transferase 3(GST3)gene and expression analysis in pearl oyster, Pinctada martensii[J]. Fish&Shellfish Immunology, 2011, 31:823-830. doi:/10.1016/j.fsi.2011.07.023.
• [4] CHEN J H, SHU X, YU Z N. F-type lectin involved in defense against bacterial infection in the pearl oyster(Pinctada martensii)[J]. Fish&Shellfish Immunology,2011, 30:750-754. doi:/10.1016/j.fsi.2010.12.025.
• [5] WANG Q H, JIAO Y, DU X D, et al. Molecular characterization and expression analysis of purple acid phosphatase gene from pearl oyster Pinctada martensii[J].Genetics and Molecular Research:GMR, 2015, 14:552-562. doi:/10.4238/2015.January.26.10.
• [6] JIAO Y, TIAN Q L, DU X D, et al. Molecular characterization of tumor necrosis factor receptor-associated factor 6(TRAF6)in pearl oyster Pinctada martensii[J]. Genetics and Molecular Research:GMR, 2014, 13:10545-10555. doi:/10.4238/2014.December.12.17.
• [7]焦钰,杜晓东,王庆恒,等.马氏珠母贝IKKε同源基因的克隆及表达分析[J].基因组学与应用生物学, 2014,33(2):266-272. doi:/10.13417/j.gab.033.000266.
• [8]师尚丽,杜晓东,焦钰,等.马氏珠母贝TLR2基因c DNA的分子特征及组织表达分析[J].中国农学通报,2014, 30(20):35-41.
• [9] KINOSHITA S, WANG N, INOUE H, et al. Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster[J]. PloS One,2011, 6:e21238. doi:/10.1371/journal.pone.0021238.
• [10] ZHENG Z, LIANG J L, HUANG R L, et al.Identification of a novel mi R-146a from Pinctada martensii involved in the regulation of the inflammatory response[J]. Fish&Shellfish Immunology, 2016, 54:40-45.
• [11] SUZUKI M M, BIRD A. DNA methylation landscapes:provocative insights from epigenomics[J]. Nature Reviews Genetics, 2008, 9:465-476. doi:/10.1038/nrg2341.
• [12] LISTER R, PELIZZOLA M, DOWEN R H, et al.Human DNA methylomes at base resolution show widespread epigenomic differences[J]. Nature, 2009,462:315-322. doi:/10.1038/nature08514.
• [13] BOFFELLI D, TAKAYAMA S, MARTIN D I. Now you see it:genome methylation makes a comeback in Drosophila[J]. Bio Essays:News and Reviews in Molecular, Cellular and Developmental Biology, 2014,36:1138-1144. doi:/10.1002/bies.201400097.
• [14] XIANG H, ZHU J D, CHEN Q, et al. Single base-resolution methylome of the silkworm reveals a sparse epigenomic map[J]. Nature Biotechnology, 2010,28:516-520.
• [15] RIVIERE G, WU G C, FELLOUS A, et al. DNA methylation is crucial for the early development in the Oyster C. gigas[J]. Marine Biotechnology(New York,NY), 2013, 15:739-753. doi:/10.1007/s10126-013-9523-2.
• [16] GAVERY M R, ROBERTS S B. DNA methylation patterns provide insight into epigenetic regulation in the Pacific oyster(Crassostrea gigas)[J]. BMC Genomics,2010, 11:483. doi:/10.1186/1471-2164-11-483.
• [17] WANG X T, LI Q Y, LIAN J M, et al. Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation[J]. BMC Genomics, 2014, 15:1119. doi:/10.1186/1471-2164-15-1119.
• [18] REYNA-LóPEZ G E, SIMPSON J, RUIZ-HERRERA J.Differences in DNA methylation patterns are detectable during the dimorphic transition of fungi by amplification of restriction polymorphisms[J]. Molecular Genetics and Genomics, 1997, 253(6):703-710.
• [19] MCCLELLAND M, NELSON M, RASCHKE E. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases[J]. Nucleic Acids Research, 1994, 22(17):3640-3659.
• [20] CHEN X Q, MA Y, CHEN F, et al. Analysis of DNA methylation patterns of PLBs derived from Cymbidium hybridium based on MSAP[J]. Plant Cell Tissue and Organ Culture, 2009, 98(1):67-77.
• [21] CHAKRABARTY D, YU K W, PAEK K Y. Detection of DNA methylation changes during somatic embryogenesis of Siberian ginseng(Eleuterococcus senticosus)[J]. Plant Science(Oxford), 2003, 165(1):61-68.
• [22]于涛,杨爱国,吴彪,等.栉孔扇贝、虾夷扇贝及其杂交子代的MSAP分析[J].水产学报, 2010, 34(9):1335-1342.
• [23]姜群.长牡蛎DNA甲基化研究[D].青岛:中国海洋大学, 2015.
• [24]贺彦斌,台培东,孙梨宗,等.稀土元素铈对斑马鱼肝脏的遗传毒性[J].生态学杂志, 2018, 37(9):2786-2793.
• [25]李炎璐,陈超,陈建国,等.云纹石斑鱼、鞍带石斑鱼及其杂交F_1的DNA甲基化分析[J].渔业科学进展,2019, 40(6):98-104.
• [26]罗少杰,邓岳文,郑哲,等.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.
• [27] DU X D, FAN G Y, JIAO Y, et al. The pearl oyster Pincada fucata martensii genome and multi-omic analyses provide insights into biomineralization.Gigascience, 2017, 6:1-12.
• [28]陆光远,伍晓明,陈碧云,等.油菜种子萌发过程中DNA甲基化的MSAP分析[J].科学通报, 2005, 50(24):2750-2756.
• [29]罗少杰,张鹏飞,焦钰,等.马氏珠母贝近交与杂交家系的DNA甲基化多态性比较[J].广东海洋大学学报, 2016, 36(6):9-15. doi:/10.3969/j.issn.1673-9159.2016.06.002.
• [30]孙敬锋,吴信忠.贝类血细胞及其免疫功能研究进展[J].水生生物学报, 2006, 30(5):601-607.
• [31]鹿蒙蒙.长牡蛎干扰素调节因子（Cg IRF-1和Cg IRF-8）对干扰素系统调控机制的初步研究[D].大连:大连海洋大学, 2018.
• [32] K?HLER G A, BRENOT A, HAAS-STAPLETON E, et al. Phospholipase A2 and phospholipase B activities in fungi[J]. Biochimica et Biophysica Acta(BBA)–Molecular and Cell Biology of Lipids, 2006, 1761:1391-1399. https://doi.org/10.1016/j.bbalip.2006.09.011.