王琦,赵斌,胡炜,姚琳琳,韩莎,李成林,时迪

• 1:山东省海洋科学研究院/山东省智慧海洋牧场重点实验室(筹)
• 2:清华大学科研院

摘要(Abstract)：

【目的】研究刺参（Apostichopus japonicus）在高温和低盐胁迫下的分子响应机制，为刺参抗逆品系的选育提供理论依据。【方法】将体质量（48.0±6.7）g刺参分为对照组（16℃，盐度30）、高温组（30℃，盐度30）、低盐组（16℃，盐度20）和高温低盐组（30℃，盐度20）进行2 d的实验，通过转录组测序技术对刺参体壁进行差异表达基因（DEGs）分析，然后对DEGs进行富集分析。随机挑选6个差异表达基因，采用实时荧光定量逆转录聚合酶链反应（qRT-PCR）对其进行验证。【结果】刺参体壁转录组测序获得高质量数据（clean reads）共616 880 106，各样品Q_(30)碱基占比均不低于94.91%；高温低盐组在胁迫前后筛选出4 089个差异基因，高温组胁迫前后筛选出1 907个差异基因，低盐组在胁迫前后筛选出1 275个差异基因。热休克蛋白家族中的多个蛋白（HSP90、HSP70）在高温胁迫下相较于对照组均显著上调（P <0.05）。京都基因与基因组百科全书（KEGG）富集结果显示，3个实验组与对照组的差异基因主要富集在PI3K-Akt信号通路、细胞外基质与细胞表面受体的相互作用、细胞焦点黏附等抗凋亡及免疫防御相关的通路；低盐组与对照组差异基因主要富集在脂肪酸代谢、信号传导等相关通路。qRT-PCR结果与转录组结果相一致。【结论】高温、低盐等会引起刺参的脂肪酸代谢紊乱、引发炎症反应，甚至死亡。抗凋亡蛋白基因、热休克蛋白家族基因的上调对缓解高温与低盐胁迫下的应激反应有积极作用。

关键词(KeyWords)： 刺参;生态环境;高温胁迫;低盐胁迫;差异基因

Abstract:

Keywords:

基金项目(Foundation): 山东省重点研发计划（2023LZGC019）;山东省重点研发计划（2022CXGC020412）;; 山东省农业重大技术协同推广计划（SDNYXTTG-2024-31）;; 山东省现代农业产业技术体系刺参产业技术体系（SDAIT-22）

作者(Author): 王琦,赵斌,胡炜,姚琳琳,韩莎,李成林,时迪

参考文献(References)：

• [1]廖玉麟.中国动物志:棘皮动物门:海参纲[M].北京:科学出版社, 1997.
• [2]胡炜,李成林,韩莎,等.异常气候和环境对刺参养殖业的影响及应对策略[J].海洋科学, 2018, 42(2):159-166.
• [3] ZHAO Y, YANG H S, STOREY K B, et al. RNA-seq dependent transcriptional analysis unveils gene expression profile in the intestine of sea cucumber Apostichopus japonicus during aestivation[J]. Comparative Biochemistry and Physiology Part D:Genomics and Proteomics, 2014,10:30-43.
• [4] LI Y X, KIKUCHI M, LI X Y, et al. Weighted gene c o-expression network analysis reveals potential genes involved in early metamorphosis process in sea cucumber Apostichopus japonicus[J]. Biochemical and Biophysical Research Communications, 2018, 495(1):1395-1402.
• [5] XING L L, SUN L N, LIU S L, et al. De Novo assembly and comparative transcriptome analyses of purple and green morphs of Apostichopus japonicus during body wall pigmentation process[J]. Comparative Biochemistry and Physiology Part D:Genomics and Proteomics, 2018, 28:151-161.
• [6] CHENG X C, ZHANG L B, GAO Z M, et al. Transcriptomic analysis reveals the immune response mechanisms of sea cucumber Apostichopus japonicus under noise stress from offshore wind turbine[J]. Science of The Total Environment, 2024, 906:167802.
• [7] LI C L, ZHAO W, QIN C X, et al. Comparative transcriptome analysis reveals changes in gene expression in sea cucumber(Holothuria leucospilota)in response to acute temperature stress[J]. Comparative Biochemistry and Physiology Part D:Genomics and Proteomics, 2021, 40:100883.
• [8] LIANG L Y, CHEN J W, LI Y N, et al. Insights into high-pressure acclimation:comparative transcriptome analysis of sea cucumber Apostichopus japonicus at different hydrostatic pressure exposures[J]. BMC Genomics, 2020, 21(1):68.
• [9] LI C, FANG H H, XU D X. Effect of seasonal high temperature on the immune response in Apostichopus japonicus by transcriptome analysis[J]. Fish&Shellfish Immunology, 2019, 92:765-771.
• [10] PéREZ-PORTELA R, RIESGO A, WANGENSTEEN O S, et al. Enjoying the warming Mediterranean:Transcriptomic responses to temperature changes of a thermophilous keystone species in benthic communities[J]. Molecular Ecology, 2020, 29(17):3299-3315.
• [11] XU D X, SUN L N, LIU S L, et al. Understanding the heat shock response in the sea cucumber Apostichopus japonicus,using iTRAQ-based proteomics[J]. International Journal of Molecular Sciences, 2016, 17(2):150.
• [12]商艳鹏,田燚,李晓雨,等.仿刺参7个盐度相关基因在低盐胁迫下的表达模式[J].中国农业科技导报, 2018, 20(11):145-153.
• [13]孙振兴,陈书秀,陈静,等.四种重金属对刺参幼参的急性致毒效应[J].海洋通报, 2007, 26(5):80-85.
• [14] MUNDY P C, JEFFRIES K M, FANGUE N A, et al.Differential regulation of select osmoregulatory genes and Na+/K+-ATPase paralogs may contribute to population differences in salinity tolerance in a semi-anadromous fish[J]. Comparative Biochemistry and Physiology Part A,Molecular&Integrative Physiology, 2020, 240:110584.
• [15] GUSHCHINA D Y, KALINOVSKAYA M V, MATVEEVA T A. Effects of the Pacific decadal oscillation on the characteristics of two types of El Ni?o under possible climate change[J]. Russian Meteorology and Hydrology,2020, 45(10):683-693.
• [16] HUANG L X, LIU W J, JIANG Q L, et al. Integration of transcriptomic and proteomic approaches reveals the temperature-dependent virulence of Pseudomonas plecoglossicida[J]. Frontiers in Cellular and Infection Microbiology, 2018, 8:207.
• [17] YE H, LIN Q S, LUO H. Applications of transcriptomics and proteomics in understanding fish immunity[J]. Fish&Shellfish Immunology, 2018, 77:319-327.
• [18] AVARRE J C, DUGUéR, ALONSO P, et al. Analysis of the black-chinned tilapia Sarotherodon melanotheron heudelotii reproducing under a wide range of salinities:from RNA-seq to candidate genes[J]. Molecular Ecology Resources, 2014, 14(1):139-149.
• [19] CHAPMAN R W, READING B J, SULLIVAN C V.Ovary transcriptome profiling via artificial intelligence reveals a transcriptomic fingerprint predicting egg quality in striped bass, Morone saxatilis[J]. PLoS One, 2014, 9(5):e96818.
• [20] CHUEH T C, HSU L S, KAO C M, et al. Transcriptome analysis of zebrafish embryos exposed to deltamethrin[J].Environmental Toxicology, 2017, 32(5):1548-1557.
• [21] COLLI-DULA R C, FANG X F, MORAGA-AMADOR D, et al. Transcriptome analysis reveals novel insights into the response of low-dose benzo(a)Pyrene exposure in male Tilapia[J]. Aquatic Toxicology, 2018, 201:162-173.
• [22] ROBLEDO D, FERNáNDEZ C, HERMIDA M, et al.Integrative transcriptome, genome and quantitative trait loci resources identify single nucleotide polymorphisms in candidate genes for growth traits in turbot[J]. International Journal of Molecular Sciences, 2016, 17(2):243.
• [23] SUN S M, XUAN F J, GE X P, et al. Identification of differentially expressed genes in hepatopancreas of oriental river prawn, Macrobrachium nipponense exposed to environmental hypoxia[J]. Gene, 2014, 534(2):298-306.
• [24]林跃辉,王敏. PI3-K-AKT信号转导途径与凋亡的关系[J].国际病理科学与临床杂志, 2005, 25(4):307-310.
• [25] DEROUET M, THOMAS L, CROSS A, et al. Granulocyte macrophage colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1[J]. Journal of Biological Chemistry, 2004, 279(26):26915-26921.
• [26] LI C H, YEN C H, CHEN M-Y, et al. Abstract 2234:activation of PI3K pathway by P-Rex2 is inhibited by GNMT[J]. Cancer Research, 2012, 72(8_Supplement):2234.
• [27]张令强.抑癌蛋白pten的去泛素化酶otud3转基因小鼠模型的建立及表型分析[D].北京:中国人民解放军军事医学科学院, 2015.
• [28] BADYLAK S F. The extracellular matrix as a scaffold for tissue reconstruction[J]. Seminars in Cell&Developmental Biology, 2002, 13(5):377-383.
• [29] FRANTZ C, STEWART K M, WEAVER V M. The extracellular matrix at a glance[J]. Journal of Cell Science,2010, 123(24):4195-4200.
• [30]巴华忠.细胞外基质相关蛋白表皮生长因子和肌腱蛋白在仿刺参再生过程中的表达分析[D].大连:辽宁师范大学, 2015.
• [31] QUI?ONES J L, ROSA R, RUIZ D L, et al. Extracellular matrix remodeling and metalloproteinase involvement during intestine regeneration in the sea cucumber Holothuria glaberrima[J]. Developmental Biology, 2002, 250(1):181-197.
• [32] LI Y J, HUANG J Q, LIU Z, et al. Transcriptome analysis provides insights into hepatic responses to moderate heat stress in the rainbow trout(Oncorhynchus mykiss)[J].Gene, 2017, 619:1-9.
• [33]王玲玲.贝类神经内分泌系统对免疫应答的调节机制[J].大连海洋大学学报, 2022, 37(3):363-375.
• [34] MAY M A, BISHOP K D, RAWSON P D. NMR profiling of metabolites in larval and juvenile blue mussels(Mytilus edulis)under ambient and low salinity conditions[J].Metabolites, 2017, 7(3):33.
• [35] TSENG Y C, HWANG P P. Some insights into energy metabolism for osmoregulation in fish[J]. Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology, 2008, 148(4):419-429.
• [36] DI MARTINO C, DELFINE S, PIZZUTO R, et al. Free amino acids and glycine betaine in leaf osmoregulation of spinach responding to increasing salt stress[J]. New Phytologist, 2003, 158(3):455-463.
• [37] MéNDEZ-LUCAS A, DUARTE J A G, SUNNY N E,et al. PEPCK-M expression in mouse liver potentiates,not replaces, PEPCK-C mediated gluconeogenesis[J].Journal of Hepatology, 2013, 59(1):105-113.
• [38] COADY M J, CHANG M H, CHARRON F M, et al. The human tumour suppressor gene SLC5A8 expresses a Na+–monocarboxylate cotransporter[J]. The Journal of Physiology,2004, 557(3):719-731.
• [39] GANAPATHY V, GOPAL E, MIYAUCHI S, et al. Biological functions of SLC5A8, a candidate tumour suppressor[J].Biochemical Society Transactions, 2005, 33(1):237-240.
• [40] MIYAUCHI S, GOPAL E, FEI Y J, et al. Functional identification of SLC5A8, a tumor suppressor down-regulated in colon cancer, as a Na+-coupled transporter for short-chain fatty acids[J]. Journal of Biological Chemistry, 2004, 279(14):13293-13296.