首页 >  渔业科学进展 >  刺参响应灿烂弧菌侵染的基因组DNA甲基化水平和转录组差异及其关联分析

2022, 45(3): 176-185. doi: 10.19663/j.issn2095-9869.20210424001

刺参响应灿烂弧菌侵染的基因组DNA甲基化水平和转录组差异及其关联分析

1. 上海海洋大学水产与生命学院 上海 201306;

2. 中国水产科学研究院黄海水产研究所 农业农村部海洋渔业可持续发展重点实验室 山东 青岛 266071;

3. 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 山东 青岛 266071;

4. 青岛瑞滋集团有限公司 山东 青岛 266408

收稿日期:2021-04-24
修回日期:2021-04-27

基金项目:   国家重点研发计划(2018YFD0901603)  山东省农业良种工程课题(2020LZGC015)  中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金(2020TD40  2021GH05)共同资助 

关键词: 刺参 , DNA甲基化 , 转录组 , 关联分析

Genomic DNA Methylation Levels and Transcriptome Differences of Apostichopus japonicus in Response to Vibrio splendidus Infection and Their Association Analysis

1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;

2. Key Laboratory of Sustainable and Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266071, China;

3. Pilot National Laboratory for Marine Science and Technology (Qingdao), Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao, Shandong 266071, China;

4. Qingdao Ruizi Company, Qingdao, Shandong 266408, China

Received Date:2021-04-24
Accepted Date:2021-04-27

Keywords: Apostichopus japonicus , DNA methylation , Transcriptome , Association analysis

为探讨病原菌胁迫下刺参(Apostichopus japonicus)基因组DNA甲基化水平和转录组表达的差异,本研究采用人工攻毒侵染胁迫,获得刺参化皮体壁组织,并以未攻毒组的健康体壁组织为对照,对刺参2种体壁组织进行全基因组甲基化测序(WGBS)和转录组高通量测序,解析刺参体壁基因组DNA甲基化差异,筛选响应病原胁迫的差异甲基化区域和差异表达基因。同时,通过基因组甲基化和转录组联合分析,筛选负相关关联基因,为解析刺参响应灿烂弧菌(Vibrio splendidus)侵染的分子机理提供基础数据。结果显示,刺参对照组和侵染组基因组总甲基化水平分别为(3.59±0.04)%和(3.87±0.27)%,侵染组甲基化水平显著升高;在发生甲基化的位点中,攻毒组和对照组的mCpG占比分别为83.06%和81.91%,mCpG为最主要的甲基化形式。本研究共筛选出差异甲基化区域(DMRs) 626 677个,注释到23 706个功能基因。转录组测序共检测到29 290个基因,筛选到差异表达基因496个,其中,上调基因214个,下调基因282个。在基因组甲基化与转录组的关联分析中,筛选到180个负相关关联基因,其中,差异甲基化区域位于启动子区域的负相关基因为60个。对负相关关联基因的GO和KEGG富集分析,筛选到相关通路和LRRhsp20CARD等关键基因。本研究将为解析刺参抗病的表观遗传调控机制提供数据,也为刺参抗病性状选育提供科学参考。

DNA methylation is an important epigenetic modification that plays a key role in gene expression regulation. In this study, two groups of sea cucumbers (Apostichopus japonicus) were prepared. One group had skin ulceration syndrome body wall (PT16S) under stress from Vibrio splendidus infection at a concentration of 1×106CFU/mL (LD50); the other group had a healthy body wall (PT10H). Genomic DNA methylation levels and gene expression differences between the two groups were detected using whole-genome bisulfite sequencing (WGBS) and transcriptome sequencing. The key gene ontology (GO) terms and KEGG terms engaged in the immune response were selected using association analysis. The results showed that the total methylation levels of the A. japonicus genome of PT10H and PT16S were (3.59±0.04)% and (3.87±0.27)%, respectively. The methylation levels of the A. japonicus genome under pathogen challenge significantly increased; mCpG accounted for 83.06% and 81.91% of all the methylated sites in PT16S and PT10H, respectively, indicating that mCpG was the most important methylation form in the sea cucumber. A total of 626 677 differentially methylated regions (DMRs) were screened and annotated into 23 706 functional genes. A total of 496 differentially expressed genes were screened, including 214 up-regulated and 282 down-regulated genes. A total of 180 negatively correlated genes were isolated using association analysis between genomic methylation and transcriptome, of which 60 genes had DMRs located in promoter regions. Based on GO and KEGG enrichment of the 180 negatively correlated genes, key genes such as LRR, Hsp20, and CARD were selected to play a critical role in the immune response. The results would provide primary data for the epigenetic regulatory mechanisms of A. japonicus and provide a theoretical reference for A. japonicus breeding.

参考文献

[1] BEG A A, BALDWIN A S. Activation of multiple NF-kappa B/Rel DNA-binding complexes by tumor necrosis factor. Oncogene, 1994, 9(5):1487-1492
[2] BIRD A. DNA methylation patterns and epigenetic memory. Genes and Development, 2002, 16(1):6-21
[3] 曹哲明, 杨健. 背角无齿蚌不同组织的基因组DNA甲基化分析. 生态环境学报, 2009, 18(6):2011-2016CAO Z M, YANG J. Analysis of the methylation in genome DNA from different tissues of Anodonta woodiana. Ecology and Environmental Sciences, 2009, 18(6):2011-2016
[4] CHOY M K, MOVASSAGH M, GOH H, et al. Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated. BMC Genomics, 2010, 11(1):519
[5] 高杉, 杨爱馥, 董颖, 等. 仿刺参"化皮病"体壁组织DNA甲基化的MSAP分析. 水生生物学报, 2017, 41(3):637-642GAO S, YANG A F, DONG Y, et al. MSAP analysis of DNA methylation in the body wall of Apostichopus japonicus. Acta Hydrobiologica Sinica, 2017, 41(3):637-642
[6] GOU X P, HE K, YANG H, et al. Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana. BMC Genomics, 2010, 11:19
[7] 郭婷婷. 刺参不同组织基因组DNA甲基化状态MSAP分析及HPLC方法的建立. 上海海洋大学硕士研究生学位论文, 2013GUO T T. The method to assess genome DNA methylation of Apostichopus japonicus by HPLC and MSAP analysis in different tissues. Masterxs Thesis of Shanghai Ocean University, 2013
[8] HAN L S, SUN Y, CAO Y, et al. Analysis of the gene transcription patterns and DNA methylation characteristics of triploid sea cucumbers (Apostichopus japonicus). Scientific Reports, 2021, 11(1):7564
[9] KRUEGER F, ANDREWS S R. Bismark:A flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics, 2011, 27(11):1571-1572
[10] LI J K, WU X W, TAN J, et al. Molecular cloning of the heat shock protein 20 gene from Paphia textile and its expression in response to heat shock. Chinese Journal of Oceanology and Limnology, 2015, 33(4):919-927
[11] 李尚俊, 孙国华, 李雪燕, 等. 高温胁迫下仿刺参表观遗传调控相关基因的表达特征. 中国水产科学, 2017, 24(3):470-476LI S J, SUN G H, LI X Y, et al. Characteristics of epigenetic regulation of related genes under high temperature stress in sea cucumber Apostichopus japonicus. Journal of Fishery Sciences of China, 2017, 24(3):470-476
[12] 李晓妮. 刺参肠道再生的DNA甲基化调控解析及再生候选基因的功能分析. 中国科学院大学(中国科学院海洋研究所)博士研究生学位论文, 2018LI X N. Analysis of DNA methylation regulation and function of candidated genes during intestinal regeneration in the sea cucumber Apostichopus japonicus. Doctoral Dissertation of Institute of Oceanology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 2018
[13] 李炎璐, 陈超, 陈建国, 等. 云纹石斑鱼、鞍带石斑鱼及其杂交F1的DNA甲基化分析. 渔业科学进展, 2019, 40(6):98-104LI Y L, CHEN C, CHEN J G, et al. DNA methylation analysis of Epinephelus moara, Epinephelus lanceolatus and their F1 hybrid. Progress in Fishery Sciences, 2019, 40(6):98-104
[14] 李玉强, 王睿甲, 李语丽, 等. 基于MethylRAD-Seq技术对仿刺参DNA甲基化图谱的研究. 中国海洋大学学报(自然科学版), 2018, 48(9):41-50LI Y Q, WANG R J, LI Y L, et al. Genome-wide profiling of DNA methylation in Apostichopus japonicus based on methylRAD-Seq. Periodical of Ocean University of China (Natural Science), 2018, 48(9):41-50
[15] LI Y, WANG R, XUN X, et al. Sea cucumber genome provides insights into saponin biosynthesis and aestivation regulation. Cell Discovery, 2018, 4(1):29
[16] MOORE L D, LE T, FAN G P. DNA methylation and its basic function. Neuropsychopharmacology, 2013, 38(1):23-38
[17] SUN H J, ZHOU Z C, DONG Y, et al. Insights into the DNA methylation of sea cucumber Apostichopus japonicus in response to skin ulceration syndrome infection. Fish and Shellfish Immunology, 2020, 104:155-164
[18] SUN Y, HOU R, FU X, et al. Genome-wide analysis of DNA methylation in five tissues of Zhikong scallop, Chlamys farreri. PloS One, 2014, 9(1):e86232
[19] TOSI L, ANIELLO F, GERACI G, et al. DNA methyltransferase activity in the early stages of a sea urchin embryo-evidence of differential control. FEBS Letters, 1995, 361(1):115-117
[20] TWEEDIE S, CHARLTON J, CLARK V, et al. Methylation of genomes and genes at the invertebrate-vertebrate boundary. Molecular and Cellular Biology, 1997, 17(3):1469-1475
[21] 王印庚, 荣小军, 廖梅杰, 等. 刺参健康养殖与病害防控技术丛解. 北京:中国农业出版社, 2014WANG Y G, RONG X J, LIAO M J, et al. Sea cucumber culture and disease control technology. Beijing:China Agriculture Press, 2014
[22] 魏书磊. 斑马鱼(Danio rerio)重组CD36基因的原核表达及功能研究. 中国海洋大学硕士研究生学位论文, 2013WEI S L. Expression and functional analysis of CD36 gene in zebrafish. Master's Thesis of China Ocean University, 2013
[23] 温争争, 左闪, 陈梦, 等. 刺参基因组DNA甲基化水平及模式对温度变化的响应. 渔业科学进展, 2021, 42(3):46-54WEN Z Z, ZUO S, CHEN M, et al. DNA methylation level of genomic DNA of Apostichopus japonicus at different temperatures. Progress in Fishery Sciences, 2021, 42(3):46-54
[24] 吴彪, 杨爱国, 孙秀俊, 等. 急性温度胁迫对虾夷扇贝(Patinopecten yessoensis)基因组DNA甲基化的影响. 渔业科学进展, 2016, 37(5):140-146WU B, YANG A G, SUN X J, et al. Effects of acute temperature stress on genome-wide DNA methylation profiles in Patinopecten yessoensis. Progress in Fishery Sciences, 2016, 37(5):140-146
[25] YANG Y J, ZHENG Y Q, SUN L N, et al. Genome-wide DNA methylation signatures of sea cucumber Apostichopus japonicus during environmental induced aestivation. Genes, 2020, 11(9):1020
[26] 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. 水产学报, 2010, 34(9):1335-1342YU T, YANG A G, WU B, et al. Analysis of Chlamys farreri, Patinopecten yessoensis and their offspring using methylation-sensitive amplification polymorphism (MSAP). Journal of Fisheries of China, 2010, 34(9):1335-1342
[27] ZHANG X J, SUN L, YUAN J B, et al. The sea cucumber genome provides insights into morphological evolution and visceral regeneration. PLoS Biology, 2017, 15(10):e2003790
[28] ZHAO Y, CHEN M Y, STOREY K B, et al. DNA methylation levels analysis in four tissues of sea cucumber Apostichopus japonicus based on fluorescence-labeled methylation- sensitive amplified polymorphism (F-MSAP) during aestivation. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology, 2015, 181:26-32
[29] 赵业. 刺参Apostichopus japonicus (Selenka)夏眠期间基因表达模式及DNA甲基化基础特征研究. 中国科学院研究生院(海洋研究所)博士研究生学位论文, 2015ZHAO Y. Study on gene expression patterns and basic characteristics of DNA methylation in Apostichopus japonicus (Selenka) during aestivation. Doctoral Dissertation of Institute of Oceanology, Chinese Academy of Sciences, 2015
[30] 卓梅琴, 杨水波, 凌仕诚, 等. 饲料脂肪对黄颡鱼卵巢脂类代谢以及PI3KCa甲基化和转录水平的影响. 水产学报, 2019, 43(10):2186-2196ZHUO M Q, YANG S B, LING S C, et al. Effects of dietary lipid on lipid metabolism, methylation and expression of PI3KCa in the ovary of yellow catfish (Pelteobagrus fulvidraco). Journal of Fisheries of China, 2019, 43(10):2186-2196
[31] 左闪, 温争争, 周红学, 等. 基于MSAP技术的刺参选育群体基因组表观与序列遗传多样性分析. 渔业科学进展, 2021, 42(3):38-45ZUO S, WEN Z Z, ZHOU H X, et al. Evaluation of epigenetic and genome sequence diversity in sea cucumber Apostichopus japonicus selected population based on MSAP technology. Progress in Fishery Sciences, 2021, 42(3):38-45
[32] 左之良, 谭杰, 吴彪, 等. 普通刺参(Apostichopus japonicus)和白刺参不同组织基因组DNA的MSAP研究. 渔业科学进展, 2016, 37(3):93-100ZUO Z L, TAN J, WU B, et al. MSAP analysis of genomic DNA in the tissues of Apostichopus japonicus and white A. japonicus. Progress in Fishery Sciences, 2016, 37(3):93-100

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目录

刺参响应灿烂弧菌侵染的基因组DNA甲基化水平和转录组差异及其关联分析