Sequence Analysis and Expression Patterns of miR-10 and Target Gene arhgef3 in Sea Cucumber Apostichopus japonicus Exposed to Salinity Stress
GOU Yuqing, WEI Xin, MA Nanxing, LIU Dan, GAO Siqi, TIAN Yi
Key Laboratory of Mariculture & Stock Enhancement in North China′s Sea, Ministry of Agriculture and Rural Affairs, College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China
Abstract：In order to investigate the response process of sea cucumber Apostichopus japonicus exposed to salinity stress, differentially expressed miR-10 and the corresponding target gene arhgef3 were screened from the transcriptome and high-throughput sequencing results of miRNAs to analyze the expression of miR-10 and target gene arhgef3 in different tissues of sea cucumber with body weight of (18.55±3.40) g exposed to a salinity of 18 for 0 h, 6 h, 24 h and 48 h with control group at 30 normal salinity to provide a basis for their roles in the salinity response mechanism of the sea cucumber. Analysis of the precursor and mature sequences of miR-10 showed that miR-10 formed a stable stem-loop structure with highly conserved regions in the interval between 29 and 51. The target gene arhgef3 had an open reading frame of 1728 bp encoding 575 amino acids and a functional domain between amino acids 280—500. There was significantly higher expression level of arhgef3 in intestine, respiratory tree and coelomocytes than that in other tissues (P<0.05). Under low salt conditions, the expression level of miR-10 was significantly lower at 24 h and significantly higher at 48 h than at 0 h in the control group, and the expression level of arhgef3 was significantly higher at 6 h of stress than at 0 h in the control group. Transfection of miR-10 inhibitors into coelomocytes was found to be significantly decreased at 24 h and increased at 48 h compared with 0 h in the experimental group, with significantly higher in the experimental group at 0 h at both 6 h and 48 h after transfection with miR-10 mimics than that at 0 h. The expression level of arhgef3 in the experimental group transfected with miR-10 mimic was found to be increased significantly at 24 hours, without significant change in expression level of arhgef3 in the experimental group transfected with miR-10 inhibitor in sea cucumber. The findings indicated that arhgef3 was induced by salinity, and that the target gene was required to maintain a high expression level to adapt to salinity changes and adapt to the low salinity response of sea cucumber during the response to salinity stress.
[1] 陈勇,高峰,刘国山,等.温度、盐度和光照周期对刺参生长及行为的影响[J].水产学报,2007,31(5):687-691. [2] 于东祥,孙慧玲,陈四清,等. 海参健康养殖技术[M]. 北京:海洋出版社,2010. [3] 张彬,韦嫔媛,熊建华,等.凡纳滨对虾胚胎发育进程及诱导染色体加倍时间确定[J].南方农业学报,2014,45(4):664-670. [4] 王冲,田燚,常亚青,等.盐度胁迫对刺参非特异性免疫酶的影响[J].中国农业科技导报,2013,15(3):163-168. [5] DONG Y W, DONG S L, MENG X L. Effects of thermal and osmotic stress on growth, osmoregulation and Hsp70 in sea cucumber (Apostichopus japonicus Selenka)[J]. Aquaculture,2008,276(1/2/3/4):179-186. [6] ASHA P S, MUTHIAH P. Effects of temperature, salinity and pH on larval growth, survival and development of the sea cucumber Holothuria spinifera Theel[J]. Aquaculture,2005,250(3/4):823-829. [7] HAMEL J F, CONAND C, PAWSON D L, et al. The sea cucumber Holothuria scabra (Holothuroidea:Echinodermata):its biology and exploitation as Beche-de-mer[M]//Advances in Marine Biology. Amsterdam:Elsevier,2001:129-223. [8] YUAN X T, YANG H S, WANG L L, et al. Effects of salinity on energy budget in pond-cultured sea cucumber Apostichopus japonicus (Selenka) (Echinodermata:Holothuroidea)[J]. Aquaculture,2010,306(1/2/3/4):348-351. [9] WANG F Y, YANG H S, GAO F, et al. Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus[J]. Comparative Biochemistry and Physiology Part A:Molecular & Integrative Physiology,2008,151(4):491-498. [10] GREGORY R I, YAN K P, AMUTHAN G, et al. The microprocessor complex mediates the genesis of microRNAs[J]. Nature,2004,432(7014):235-240. [11] FARAZI T A, JURANEK S A, TUSCHL T. The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members[J]. Development,2008,135(7):1201-1214. [12] BARTEL D P. MicroRNAs:genomics, biogenesis, mechanism, and function[J]. Cell,2004,116(2):281-297. [13] HAN J J, XU G L, XU T J. The miiuy croaker microRNA transcriptome and microRNA regulation of RIG-I like receptor signaling pathway after poly(I:C) stimulation[J]. Fish & Shellfish Immunology,2016,54:419-426. [14] 覃玉蓉,夏新莉,尹伟伦.实时荧光定量PCR检测miR169g在脱水与高盐胁迫下胡杨叶中的表达[J].现代仪器,2011,17(3):28-30. [15] 赵笑颜,吕建建,张文,等.miR-139555及其靶基因PtNBC在三疣梭子蟹适应盐度胁迫中的表达调控[J].水生生物学报,2022,46(3):316-323. [16] ZHENG J B, CAO J W, MAO Y, et al. Identification of microRNAs with heat stress responsive and immune properties in Marsupenaeus japonicus based on next-generation sequencing and bioinformatics analysis:essential regulators in the heat stress-host interactions[J]. Fish & Shellfish Immunology,2018,81:390-398. [17] 裴田瑛,颜帅帅,许强华.斑马鱼miR-30e对血细胞生成的作用机制研究[J].大连海洋大学学报,2021,36(2):229-233. [18] ZHANG P J, LI C H, ZHANG R, et al. The roles of two miRNAs in regulating the immune response of sea cucumber[J]. Genetics,2015,201(4):1397-1410. [19] XIAO Y T, YAN W H, LU L N, et al. p38/p53/miR-200a-3p feedback loop promotes oxidative stress-mediated liver cell death[J]. Cell Cycle,2015,14(10):1548-1558. [20] ZHONG L, ZHANG F, ZHAI Y, et al. Identification and comparative analysis of complement C3-associated microRNAs in immune response of Apostichopus japonicus by high-throughput sequencing[J]. Scientific Reports,2015,5:17763. [21] LU M, ZHANG P J, LI C H, et al. miRNA-133 augments coelomocyte phagocytosis in bacteria-challenged Apostichopus japonicus via targeting the TLR component of IRAK-1 in vitro and in vivo[J]. Scientific Reports,2015,5:12608. [22] 尹英超,刘富祥,张玉钦,等.急性高温低氧胁迫下刺参体腔液细胞miRNA表达谱的构建和靶标基因的功能分析[J].中国海洋大学学报(自然科学版),2022,52(4):30-42. [23] 傅意然,田燚,常亚青,等.低盐胁迫对刺参4个盐度调节相关基因表达丰度的影响[J].中国水产科学,2014,21(5):902-909. [24] 郭然,吴玮杰,商艳鹏,等.Aja-miR-22和Aja-miR-27及其靶基因在刺参盐度胁迫后的表达模式[J].中国水产科学,2020,27(4):375-382. [25] 赵业,卜少阳,王涵,等.刺参甜菜碱高半胱氨酸甲基转移酶1基因(bhmt1)的克隆、分析及其在低盐胁迫下的表达变化[J].烟台大学学报(自然科学与工程版),2022,35(4):434-442. [26] MULLIN B H, MAMOTTE C, PRINCE R L, et al.Influence of ARHGEF3 and RHOA knockdown on ACTA2 and other genes in osteoblasts and osteoclasts[J].PLoS One,2014,9(5):e98116. [27] 林李泉,刘明珠,林国荣,等.黑鲷NKCC1分子特征及其对急性盐度胁迫的表达响应[J].广西科学院学报,2021,37(2):133-143. [28] 徐娴,何琳,林志华,等.盐度胁迫下缢蛏渗透压变化及V-ATPase H基因的表达分析[J].动物学杂志,2020,55(5):606-613. [29] 范红弟,李运东,杨其彬,等.斑节对虾MKK7基因的克隆及在不同胁迫条件下的表达分析[J].中国水产科学,2020,27(7):748-758. [30] 周叶,王小敏,洪一江.池蝶蚌IRAK4基因的克隆及功能[J].南昌大学学报(理科版),2021,45(4):383-393. [31] 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. [32] PNG K J, HALBERG N, YOSHIDA M, et al. A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells[J]. Nature,2011,481(7380):190-194. [33] JUNG H M, HU C T, FISTER A M, et al. MicroRNA-mediated control of developmental lymphangiogenesis[J]. eLife,2019,8:e46007. [34] YUAN J, XIAO G L, PENG G, et al. MiRNA-125a-5p inhibits glioblastoma cell proliferation and promotes cell differentiation by targeting TAZ[J]. Biochemical and Biophysical Research Communications,2015,457(2):171-176. [35] DENG D N, WANG L, CHEN Y, et al. MicroRNA-124-3p regulates cell proliferation, invasion, apoptosis, and bioenergetics by targeting PIM1 in astrocytoma[J]. Cancer Science,2016,107(7):899-907. [36] SUZUKI H I. MicroRNA control of TGF-β signaling[J]. International Journal of Molecular Sciences,2018,19(7):1901. [37] YAN B, ZHAO L H, GUO J T, et al. miR-429 regulation of osmotic stress transcription factor 1 (OSTF1) in tilapia during osmotic stress[J]. Biochemical and Biophysical Research Communications,2012,426(3):294-298. [38] 蒋亚男.3个microRNA及靶基因在刺参盐度适应中的作用[D].大连:大连海洋大学,2019. [39] 陈铭,林志华,徐娴,等.盐度胁迫对缢蛏血液离子浓度及碳酸酐酶基因表达的影响[J].生物学杂志,2021,38(1):83-87.
2024, Vol. 43 
