大口黑鲈<em>MyD88</em>基因的克隆及其对NF-κB的激活作用

doi:10.3969/j.issn.1674-7968.2025.02.011

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Abstract Adapter protein myeloid differentiation factor 88 (MyD88) is involved in the interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR)-mediated activation of nuclear factor-kappaB (NF-κB), which plays important role in innate immunity. In this study, the full-length cDNA of largemouth bass (Micropterus salmoides) MyD88 was isolated. Its ORF was 867 bp in length which encoded 288 amino acid residues. Protein secondary structure analysis showed that MyD88 protein had a N-terminal death domain and a TIR (Toll-like/IL-1) domain, which are known as important functional structural domains in mammalian MyD88. The largemouth bass MyD88 protein had high identity (58.5%~99.3%) with other vertebrates. Phylogenetic analysis showed that largemouth bass MyD88 gathered together with MyD88 of other fish species. In healthy largemouth bass, MyD88 mRNA was detected in all sampled tissues, and MyD88 mRNA had the highest expression levels in the liver. To study the role of MyD88 in innate immunity, its mRNA expression profile after stimulation with polyinosinic-polycytidylic acid (PolyI:C) and Nocardia seriolae was studied in the intestine, gill, spleen and kidney. The results showed that in all 4 detected issues, 2 kinds of immune stimuli both upregulated the transcription level of largemouth bass MyD88, and the induction by N. seriolae in the kidney was the strongest. In kidney tissue, MyD88 showed the maximum upregulation at 9 d after infection, and the expression level in the infection group was 8.5 times of that in the control group (P<0.05). The induction by PolyI:C in the gill was initiated earliest. In gill tissue, after 8 h of stimulation, the relative expression level of the experimental group was 5.6 times of that in the control group. Largemouth bass MyD88 were distributed in the HeLa cytoplasm. Overexpression of MsMyD88 could activate NF-κB. These results provide basic data for elucidating the role of MyD88 in the innate immunity of largemouth bass.

Key words： Largemouth bass (Micropterus salmoides) Myeloid differentiation factor 88 (MyD88) Cloning Expression analysis Subcellular localization Nuclear factor kappa-B (NF-κB)

Received: 12 June 2024

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S917.4

Corresponding Authors: * sunchengfei@prfri.ac.cn

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	GAO Feng-Ying
	DONG Jun-Jian
	ZHANG He-Tong
	LI Jia-Xin
	ZHU Zhi-Lin
	SUN Cheng-Fei
	Ye Xing

Cite this article:

GAO Feng-Ying,DONG Jun-Jian,ZHANG He-Tong, et al. Cloning of Largemouth Bass (Micropterus salmoides) MyD88 Gene and Its Activating Effect on NF-κB[J]. 农业生物技术学报, 2025, 33(2): 366-378.

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https://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2025.02.011 OR https://journal05.magtech.org.cn/Jwk_ny/EN/Y2025/V33/I2/366

[1] 王荣华, 李伟, 肖调义, 等. 2016. 赤眼鳟Myd88基因cDNA克隆与表达特征性研究[J]. 水生生物学报, 40(3): 459-466.
(Wang R H, Li W, Xiao T Y, et al.2016. Molecular cloning, characterization and expression of MyD88 in Squaliobarbus curriculus[J]. Acta Hydrobiologica Sinica, 40(3): 459-466.)
[2] 谢苏明, 徐钢春, 王裕玉, 等. 2021.投喂频率对池塘循环水养殖大口黑鲈消化酶、组织结构及脂代谢酶基因表达的影响[J]. 中国水产科学, 28(2): 157-166.
(Xie S M, Xu G C, Wang Y Y, et al.2021. Effects of feeding frequency on digestive enzymes, histomorphology, and gene expression of lipid metabolic enzymes of largemouth bass (Micropterus salmoides) reared in in-pond raceway culture systems[J]. Journal of Fishery Sciences of China, 28(2): 157-166.)
[3] 姚志刚, 冯建军, 王艺磊, 等. 2015. 欧洲鳗鲡Myd88基因的克隆及其免疫功能分析[J].水产学报, 39(3): 305-317.
(Yao Z G, Feng J, Wang Y, et al.2015. Molecular cloning and immune function analysis of MyD88 gene in Anguill Anguilla[J]. Journal of Fisheries of China, 39(3): 305-317.)
[4] Burns K, Janssens S, Brissoni B, et al.2003. Inhibition of interleukin 1 receptor/toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4[J]. Journal of Experimental Medicine, 197: 263-268.
[5] Camus A, Griffin M, Armwood A, et al.2019. A spontaneous outbreak of systemic Edwardsiella piscicida infection in largemouth bass Micropterus salmoides (Lac´ep`ede, 1802) in California, USA[J]. Journal of Fish Disease, 42: 759-763.
[6] Carrizo V, Valenzuela C A, Aros C, et al.2021. Transcriptomic analysis reveals a Piscirickettsia salmonis-induced early inflammatory response in rainbow trout skeletal muscle[J]. Comparative Biochemistry and Physiology Part D, Genomics & Proteomics, 39: 100859.
[7] Chen J H, Li X, Wang M J, et al.2020. Molecular cloning and expression analysis of myd88 from oriental weatherfish (Misgurnus anguillicaudatus) in response to bacterial challenge[J]. Journal of Fish Biology, 96: 1341-1348.
[8] Feng H, Su R, Song Y, et al.2016. Positive correlation between enhanced expression of TLR4/MyD88/NF-kappaB with insulin resistance in placentae of gestational diabetes mellitus[J]. PLOS ONE, 11(6): e0157185.
[9] Gao Q, Yin F, Zhang C, et al.2017. Cloning, characterization, and function of MyD88 in silvery pomfret (Pampus argenteus) in response to bacterial challenge[J]. International Journal of Biological Macromolecules, 103:327-337.
[10] Gui J F.2015. Fish biology and biotechnology is the source for sustainable aquaculture[J]. Science China Life Sciences, 58: 121-123.
[11] Guo C, Zhang L, Nie L, et al.2016. Association of polymorphisms in the MyD88, IRAK4 and TRAF6 genes and susceptibility to type 2 diabetes mellitus and diabetic nephropathy in a southern Han Chinese population[J]. Molecular and Cellular Endocrinology, 429: 114-119.
[12] Han R, Zeng Y R, Ni L Y, et al.2019. Grouper (Epinephelus coioides) MyD88 adaptor-like (Mal): Molecular cloning, expression, and functionality[J]. Fish and Shellfish Immunology, 93: 308-312.
[13] Hofmann K, Tschopp J.1995. The death domain motif found in FAS (APO-1) and TNF receptor is present in proteins involved in apoptosis and axonal guidance[J]. FEBS Letters, 371: 321-323.
[14] Hosseini R, Lamers G E M, Bos E, et al.2021.The adapter protein Myd88 plays an important role in limiting mycobacterial growth in a zebrafish model for tuberculosis[J].Virchows Archiv, 479: 265-275.
[15] Hu Q P, Mao D A.2016. Histone deacetylase inhibitor SAHA attenuates post-seizure hippocampal microglia TLR4/MYD88 signaling and inhibits TLR4 gene expression via histone acetylation[J]. BMC Neuroscience, 17(1): 22.
[16] Itoh N, Nagata S.1993. A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen[J]. Journal of Biological Chemistry, 268: 10932-10937.
[17] Janssens S, Burns K, Tschopp J, et al.2002. Regulation of interleukin-1 and lipopolysaccharide-induced NF-jB activation by alternative splicing of MyD88[J]. Current Biology, 12: 467-471
[18] Kawai T, Akira S.2010. The role of pattern-recognition receptors in innate immunity: Update on Toll-like receptors[J]. Nature Immunology, 11(5): 373-384.
[19] Krishnan J, Selvarajoo K, Tsuchiya M, et al.2007. Toll-like receptor signal transduction[J]. Experimental & Molecular Medicine, 39: 421-438.
[20] Li Y W, Wang Z, Mo Z Q, et al.2015. Grouper (Epinephelus coioides) MyD88 and Tollip: Intracellular localization and signal transduction function[J]. Fish and Shellfish Immunology, 42: 153-158.
[21] Lin J Y, Hu G B, Yu C H, et al.2015. Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus)[J]. Developmental and Comparative Immunology, 52: 166-171.
[22] Liu X, Li X, Du X, et al.2019. Spotted knifejaw (Oplegnathus punctatus) MyD88: Intracellular localization, signal transduction function and immune responses to bacterial infection[J]. Fish and Shellfish Immunology, 89:719-726.
[23] Liu Z G, Dong J J, Ke X L, et al.2022. Isolation, identification, and pathogenic characteristics of Nocardia seriolae in largemouth bass Micropterus salmoides[J]. Diseases of Aquatic Organisms, 149: 33-45.
[24] Ma D, Fan J, Tian Y, et al.2019. Selection of reference genes for quantitative real-time PCR normalisation in largemouth bass Micropterus salmoides fed on alternative diets[J]. Journal of Fish Biology, 95: 393-400.
[25] Medzhitov R, Preston-Hurlburt P, Kopp E, et al.1998. MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways[J]. Molecular Cell, 2:253-258.
[26] Qi Z T, Xu Y, Liu Y H, et al.2022. Characterization of TLR1, TLR2, TLR3, TLR5S, TLR8, TLR9, TLR21 and TLR22 of largemouth bass (Micropterus salmoides) and their expression patterns following PAMPs stimulations[J]. Aquaculture Research, 53(6): 2562-2566.
[27] Qi Z, Sun B, Zhang Q, et al.2017. Molecular cloning, structural modeling, and expression analysis of MyD88 and IRAK4 of golden pompano (Trachinotus ovatus)[J]. Developmental and Comparative Immunology, 74: 19-24.
[28] Rebl A, Goldammer T, Fischer U, et al.2009. Characterization of two key molecules of teleost innate immunity from rainbow trout (Oncorhynchus mykiss): MyD88 and SAA[J]. Veterinary Immunology and Immunopathology, 131(1-2): 122-126.
[29] Skjaeveland I, Iliev D B, Strandskog G, et al.2009. Identification and characterization of TLR8 and MyD88 homologs in Atlantic salmon (Salmo salar)[J]. Developmental Comparative Immunology, 33(9): 1011-1017.
[30] Sun C F, Li J, Dong J J, et al.2021.Chromosome-level genome assembly for the largemouth bass Micropterus salmoides provides insights into adaptation to fresh and brackish water[J]. Molecular Ecology Resources, 21(1): 301-303.
[31] Takano T, Kondo H, Hirono I, et al.2006. Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA and gene in Japanese flounder, Paralichthys olivaceus[J]. Developmental and Comparative Immunology, 30(9): 807-816.
[32] Tang D, Gao Y, Wang R, et al.2012. Characterization, genomic organization, and expression profiles of MyD88, a key adaptor molecule in the TLR signaling pathways in miiuy croaker (Miichthys miiuy)[J]. Fish Physiological Biochemistry, 38(6): 1667-1677.
[33] Tang X, Yang M, Liu J, et al.2022. Identification, functional characterization and expression pattern of myeloid differentiation factor 88 (MyD88) in Nibea albiflora[J]. Fish and Shellfish Immunology, 124: 380-390.
[34] Thorburn A N, Tseng H Y, Donovan C, et al.2016. TLR2, TLR4 AND MyD88 mediate allergic airway disease (AAD) and streptococcus pneumoniae-induced suppression of AAD[J]. PLOS ONE, 11(6): e0156402.
[35] Weber C H, Vincenz C.2001. The death domain superfamily: A tale of two interfaces?[J]. Trends in Biochemical Sciences, 26: 475-481.
[36] Whang I, Lee Y, Kim H, et al.2011. Characterization and expression analysis of the myeloid differentiation factor 88 (MyD88) in rock bream Oplegnathus fasciatus[J]. Molecular Biology Reports, 38(6): 3911-3920.
[37] Wu C, Deng H, Li D, et al.2021. Ctenopharyngodon idella Tollip regulates MyD88-induced NF-κB activation[J]. Developmental and Comparative Immunology, 123:104162.
[38] Xu Y, Tao X, Shen B, et al.2000. Structural basis for signal transduction by the Toll/inter-leukin-1 receptor domains[J]. Nature, 408: 111-115.
[39] Yuan Y, Shi Z, Wang Q, et al.2023. Molecular characterization and expression analyses of five genes involved in the MyD88-dependent pathway of yellow catfish (Pelteobagrus fulvidraco) responding to challenge of Aeromonas hydrophila[J]. Fish and Shellfish Immunology, 137: 108712.
[40] Zhang J, Kong X H, Zhou C J, et al.2014. Toll-like receptor recognition of bacteria in fish: Ligand specificity and signal pathways[J]. Fish and Shellfish Immunology, 41(2): 380-388.
[41] Zhang J, Zhu Y, Chen Z, et al.2019. Molecular cloning and expression analysis of MyD88 and TRAF6 in Qihe crucian carp Carassius auratus[J]. Fish and Shellfish Immunology, 87: 829-838.
[42] Zhou Z, Ding S, H Y, et al.2019. Northeast Chinese lamprey (Lethenteron morii) MyD88: Identification, expression, and functional characterization[J]. Fish and Shellfish Immunology, 94: 539-547.