Cloning and expression analysis of IKKβ gene in Penaeus japonicus
-
摘要: 【目的】明确日本对虾NF-κB抑制蛋白激酶β基因（PjIKKβ）生物学特征及其在各组织和哈维弧菌刺激下的表达模式，为揭示IKKβ在日本对虾先天免疫中的作用机制提供理论依据。【方法】克隆PjIKKβ基因cDNA序列，通过ORF Finder、ExPASy ProtParam、SMART、SignalP 5.0、TMHMM、Prot-Comp等在线软件进行生物信息学分析，并以实时荧光定量PCR检测PjIKKβ基因在日本对虾各组织及哈维弧菌悬液刺激后的表达模式。【结果】 PjIKKβ基因序列全长2794 bp，其开放阅读框（ORF）长2373 bp，共编码790个氨基酸残基。PjIKKβ蛋白包含1个丝氨酸/苏氨酸蛋白激酶催化结构域（S-TKc）、1个亮氨酸锌指结构域（LZ）和1个螺旋—环—螺旋结构域（HLH），定位于细胞质和细胞核中，无跨膜结构，无信号肽。PjIKKβ与凡纳滨对虾IKKβ氨基酸序列相似性最高（98%），其次是与斑节对虾IKKβ氨基酸序列（97%）；多序列比对分析结果显示，各物种IKKβ蛋白序列在S-TKc结构域的保守性较高，而在其他区域的保守性低。基于IKKβ氨基酸序列相似性构建的系统发育进化树显示，在甲壳动物分支中日本对虾先与凡纳滨对虾聚在一起，再与斑节对虾聚为一个分支。PjIKKβ基因在日本对虾肝胰腺、肌肉、鳃、神经节、胃、心脏、血淋巴和眼柄等8个组织中均有表达，以神经节、肌肉、血淋巴和眼柄中的相对表达量较高，显著高于在其他组织中的相对表达量（P＜0.05）；注射后24、48和72 h，PjIKKβ基因在注射哈维弧菌日本对虾血淋巴中的相对表达量上调，且与注射PBS日本对虾的相对表达量存在极显著差异（P＜0.01）。【结论】 PjIKKβ蛋白含有1个S-TKc结构域、1个LZ结构域和1个HLH结构域，在进化过程中非常保守；PjIKKβ基因的组织表达特征及其在哈维弧菌刺激后的表达变化，进一步证实PjIKKβ基因参与了日本对虾的先天免疫应答反应。Abstract: 【Objective】 To elucidate the biological characteristics of NF-κB inhibitory protein kinase-β gene（named PjIKKβ）in Penaeus japonicus and its expression pattern in various tissues and stimulated by Vibrio harveyi ，and to provide theoretical basis for revealing the mechanism of IKKβ in the innate immunity of P. japonicus.【Method】The cDNA sequence of PjIKKβ gene was cloned. The bioinformatics of PjIKKβ gene was analyzed by some online softwares，such as ORF Finder，ExPASy ProtParam，SMART，SignalP 5.0，TMHMM and Prot-Comp. The expression of PjIKKβ gene was examinated in various tissues under normal condition and in haemolymph after the treatment with V. harveyi by real-time fluorescence quantitative PCR.【Result】 The full length of PjIKKβ gene sequence was 2794 bp and the open reading frame （ORF）was 2373 bp which encoding 790 amino acid residues. PjIKKβ protein contained a serine/threonine protein kinases catalytic（S-TKc）domain，a leucine zipper（LZ）domain and a helix loop helix（HLH）domain. PjIKKβ protein was localized in cytoplasm and nucleus. No transmembrane structure and signal peptide were detected in PjIKKβ protein. PjKKβ protein shared the highest identity with IKKβ amino acid sequence of P. vannamei（98%）and P. monodon（97%）. Multiple sequence alignment analysis showed that S-TKc domain was highly conserved among various species and other domains were low conservation. The result of phylogenetic tree constructed based on IKKβ amino acid sequence showed that P. japonicus were clustered firstly with P. vannamei and then clustered with P. vannamei. PjIKKβ gene was ubiquitously expressed in all tested tissues（hepatopancreas，muscles，gills，ganglia，stomach，heart，hemolymph and eye stalk）. The highest expression level was detected in the ganglion，muscle，haemolymph and eye stalk. The expression of PjIKKβ gene in haemolymph was up-regulated at 24，48 and 72 h after infected of V. harveyi and was extremely significantly different from that infected with PBS（P＜0.01）.【Conclusion】 PjIKKβ protein contains a S-TKc domain，a LZ domain and a HLH domain，which is conservative in evolution. The expression of PjIKKβ in various tissues and its changes of expression after stimulation by V. harveyi indicate that PjIKKβ is involved in the innate immune response of P. japonicus.
-
Keywords:
- Penaeus japonicus /
- IKKβ gene /
- Vibrio harveyi /
- expression feature /
- innate immunity
-
-
蒋魁, 徐力文, 苏友禄, 马红玲, 刘广锋, 郭志勋, 高芳, 冯娟. 2017. 两株珍珠龙趸病原性哈维弧菌(Vibrio harveyi)的分离与鉴定[J]. 生态科学, 36(6):16-24.[Jiang K, Xu L W, Su Y L, Ma H L, Liu G F, Guo Z X, Gao F, Feng J. 2017. Isolation and identification of two pathogenetic Vibrio harveyi strains from pearl gentian[J]. Ecological Science, 36(6):16-24.]doi:10.14108/j.cnki.1008-8873. 2017.06.003. 蒋魁, 徐力文, 苏友禄, 王雨, 郭志勋, 许海东, 高芳, 冯娟. 2016. 2012年~2014年南海海水养殖鱼类病原菌哈维弧菌分离株的耐药性分析[J]. 南方水产科学, 12(6):99-107.[Jiang K, Xu L W, Su Y L, Wang Y, Guo Z X, Xu H D, Gao F, Feng J. 2016. Analysis of antibiotic resistance spectrum of Vibrio harveyi strains isolated from maricultured fish in the South China Sea during 2012 -2014[J]. South China Fisheries Science, 12(6):99-107.]doi: 10.3969/j.issn.2095-0780.2016.06.013. 农业农村部渔业渔政管理局, 全国水产技术推广总站, 中国水产学会. 2021. 2020中国渔业统计年鉴[M]. 北京:中国农业出版社.[Fishery and Fishery Administration Bureau of the Ministry of Agriculture and Rural Affairs, National Fishery Technology Extension Station,China Aquatic Society. 2021. China fishery statistics yearbook 2020[M]. Beijing:China Agricultural Press.]doi: 10.43455/y.cnki.yzytn.2021.000001. Bakkar N, Wang J X, Ladner K J, Wang H T, Dahlman J M, Carathers M, Acharyya S, Rudnicki M A, Hollenbach A D, Guttridge D C. 2008. IKK/NF-kappaB regulates skeletal myogenesis via a signaling switch to inhibit differentiation and promote mitochondrial biogenesis[J]. Journal of Cell Biology, 180 (4):787-802. doi:10.1083/jcb.2007 07179.
Chen W J, Guo C J, Zhou Z C, Yuan L Q, Xiang Z M, Weng S P, Zhang Y F, Yu X Q, He J G. 2011. Molecular cloning of IKKβ from the mandarin fish Siniperca chuatsi and its up-regulation in cells by ISKNV infection[J]. Veterinary Immunology and Immunopathology, 139(1):61-66. doi: 10.1016/j.vetimm.2010.07.025.
Cowley J A. 2020. Mourilyan virus pathogenicity in kuruma shrimp(Penaeus japonicus)[J]. Journal of Fish Diseases, 43 (11):1401-1407. doi: 10.1111/jfd.13244.
He L G, Zhao Y L, Tang L, Yu X, Ye Z F, Lin H R, Zhang Y, Li S S, Lu D Q. 2020. Molecular characterization and functional analysis of IKKα in orange-spotted grouper(Epinephelus coioides)[J]. Fish & Shellfish Immunology, 101:159-167. doi: 10.1016/j.fsi.2020.03.029.
Hinz M, Scheidereit C. 2014. The IκB kinase complex in NF-κB regulation and beyond[J]. EMBO Reports, 15:46-61.doi: 10.1002/embr.201337983.
Huang B Y, Zhang L L, Xu F, Tang X Y, Li L, Wang W, Liu M K, Zhang G F. 2019. Oyster versatile IKKα/βs are involved in toll-like receptor and RIG-I-like receptor signaling for innate immune response[J]. Frontiers in Immunology, 10:1826. doi: 10.3389/fimmu.2019.01826.
Ito M, Satoh J, Hano T, Mekata T, Ito K. 2021. Immune toxicity of phenanthrene and its combined effects of white spot syndrome virus on the survival of kuruma shrimp(Penaeus Japonicus)[J]. Ecotoxicology and Environmental Safety, 208:111640. doi: 10.1016/j.ecoenv.2020.111640.
Jiang M, Tu D D, Gu W B, Zhou Y L, Zhu Q H, Guo X L, Shu M A. 2018. Identification and functional analysis of inhibitor of NF-κB kinase(IKK)from Scylla paramamosain:The first evidence of three IKKs in crab species and their expression profiles under biotic and abiotic stresses[J]. Developmental & Comparative Immunology, 84:199-212. doi: 10.1016/j.dci.2018.02.014.
Kai X, Chellappa V, Donado C, Reyon D, Sekigami Y, Ataca D, Louissaint A, Mattoo H, Joung J K, Pillai S. 2014. IκB kinase β(IKBKB)mutations in lymphomas that constitutively activate canonical nuclear factor κB(NFκB) signaling[J]. Journal of Biological Chemistry, 289 (39):26960-26972. doi: 10.1074/jbc.M114.598763.
Kumar S, Stecher G, Tamura K. 2016. MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Molecular Biology and Evolution, 33(7):1870-1874. doi: 10.1093/molbev/msw054.
Li H P, Wu X F, Chen T, Jiang X, Ren C H. 2020. Molecular characterization, inducible expression and functional analysis of an IKKβ from the tropical sea cucumber Holothuria leucospilota[J]. Fish & Shellfish Immunology, 104:622-632. doi: 10.1016/j.fsi.2020.06.024.
Li L L, Liu W J, Fan N N, Li F S, Huang B Y, Liu Q, Wang X M, Zheng Y X, Sang X X, Dong J, Wang X N, Wei L,Liu Y Q, Zhang M W, Ma J L, Chen J W, Qi Y T, Wang X T. 2022. Scallop IKK1 responds to bacterial and virusrelated pathogen stimulation and interacts with MyD88 adaptor of toll-like receptor pathway signaling[J]. Frontiers in Immunology, 13:869845. doi:10.3389/fimmu.2022. 869845.
Lim H, Lee H, Noh K, Lee S J. 2017. IKK/NF-κB-dependent satellite glia activation induces spinal cord microglia activation and neuropathic pain after nerve injury[J]. Pain, 158 (9):1666-1677. doi: 10.1097/j.pain.0000000000000959.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods, 25(4):402-408. doi: 10.1006/meth.2001.1262.
Negi G, Sharma S S. 2015. Inhibition of IκB kinase(IKK) protects against peripheral nerve dysfunction of experimental diabetes[J]. Molecular Neurobiology, 51(2):591-598. doi: 10.1007/s12035-014-8784-8.
Nhnhkorn Z, Amparyup P, Kawai T, Tassanakajon A. 2019. Penaeus monodon IKKs participate in regulation of cytokine-like system and antiviral responses of innate immune system[J]. Frontiers in Immunology, 10:1430. doi: 10.3389/fimmu.2019.01430.
Oh H, Grinberg-Bleyer Y, Liao W, Maloney D, Wang P Z, Wu Z K, Wang J G, Bhatt D M, Heise N, Schmid R M, Hayden M S, Klein U, Rabadan R, Ghosh S. 2017. An NF-κB transcription-factor-dependent lineage-specific transcriptional program promotes regulatory T cell identity and function[J]. Immunity, 47 (3):450-465. doi: 10.1016/j.immuni.2017.08.010.
Pomerantz J L, Baltimore D. 1999. NF-κB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase[J]. The EMBO Journal, 18 (23):6694-6704. doi: 10.1093/emboj/18.23.6694.
Rothwarf D M, Zandi E, Natoli G, Karin M. 1998. IKK-γ is an essential regulatory subunit of the IκB kinase complex[J]. Nature, 395:297-300. doi: 10.1038/26261.
Sun S C. 2011. Non-canonical NF-κB signaling pathway[J]. Cell Research, 21:71-85. doi: 10.1038/cr.2010.177.
Tassanakajon A,Somboonwiwat K,Supungul P,Tang S. 2013. Discovery of immune molecules and their crucial functions in shrimp immunity[J]. Fish & Shellfish Immunology, 34 (4):954-967. doi: 10.1016/j.fsi.2012.09.021.
Wang P H, Gu Z H, Wan D H, Liu B D, Huang X D, Weng S P, Yu X Q, He J G. 2013. The shrimp IKK-NF-κB signaling pathway regulates antimicrobial peptide expression and may be subverted by white spot syndrome virus to facilitate viral gene expression[J]. Cellular & Molecular Immunology, 10 (5):423-436. doi: 10.1038/cmi.2013.30.
Xu C, Li L C, Wang C Y, Jiang J Z, Li L, Zhu L H, Jin S, Jin Z H, Lee J J, Li G H, Yan G H. 2022. Effects of G-Rh2 on mast cell-mediated anaphylaxis via AKT-Nrf2/NF-κB and MAPK-Nrf2/NF-κB pathways[J]. Journal of Ginseng Research, 46(4):550-560. doi:10.1016/j. jgr. 2021. 10.001.
Zhao M M, Joy J, Zhou W Q, De S, Wood W H, Becker K G, Ji H K, Sen R J. 2018. Transcriptional outcomes and kinetic patterning of gene expression in response to NF-κB activation[J]. PLoS Biology, 16(9):e2006347. doi:10. 1371/journal.pbio.2006347.
计量
- 文章访问数: 35
- HTML全文浏览量: 2
- PDF下载量: 8
下载: