水滴伪康纤虫交替氧化酶基因(<em>AOX3</em>)的克隆及其基因家族鉴定与表达分析

doi:10.3969/j.issn.1674-7968.2026.05.015

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摘要水滴伪康纤虫(Pseudocohnilembus persalinus)是危害海水养殖动物的盾纤毛类原生动物。本研究通过克隆水滴伪康纤虫交替氧化酶(alternative oxidase, AOX)基因,在基因组水平上对其基因家族进行系统鉴定并预测其生物学功能,解析其表达特征。利用cDNA末端快速克隆(rapid amplification of cDNA ends, RACE)技术获得水滴伪康纤虫AOX3基因的全长cDNA序列;通过Pfam序列PF01786筛选鉴定水滴伪康纤虫AOXs基因家族成员,运用生物信息学方法对其家族成员的理化性质、亚细胞定位预测、基因结构、Scaffold定位、启动子区域顺式元件和系统发育关系等进行了分析,通过qRT-PCR分析水滴伪康纤虫AOXs基因家族成员在营养期与包囊期及在温度胁迫下的表达模式。结果表明,克隆得到水滴伪康纤虫AOX3基因的cDNA全长为1 091 bp (GenBank No. PV953026),其中5'-UTR长度为47 bp,3'-UTR长度为150 bp,ORF总长度为894 bp。在其全基因组中共鉴定到4个AOXs基因家族成员(分别命名为PpAOX1~PpAOX4);PpAOXs均为无信号肽的亲水性蛋白,主要定位于细胞质和线粒体中;启动子顺式作用元件分析显示,PpAOXs可能与响应缺氧诱导、温度和防御应激等胁迫相关;对13个原生动物物种的33个AOXs家族基因进行的系统发育分析显示,水滴伪康纤虫的4个AOXs基因家族成员与寡膜纲类群形成一个支系;qRT-PCR结果显示,PpAOX1在水滴伪康纤虫的营养期表达量较高,而PpAOX2~PpAOX4的表达则在包囊期呈现显著上调;PpAOXs家族成员在应对温度胁迫时,呈现不同的表达模式。本研究为进一步挖掘PpAOXs基因的生物学功能,深入阐释水滴伪康纤虫对逆境胁迫的响应策略提供重要参考资料。

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关键词 ：水滴伪康纤虫, 交替氧化酶(AOX), 基因克隆, 基因家族, 表达分析

Abstract：Pseudocohnilembus persalinus is a pathogenic scuticociliate parasite in mariculture fish species. In this study, the alternative oxidase (AOX) gene from Pseudocohnilembus persalinus was cloned. The AOX gene family was systematically identified at the genomic level, its biological functions and expression characteristics were predicted and analyzed. The full-length cDNA of the alternative oxidase 3 (AOX3) gene from P. persalinus was cloned using the rapid amplification of cDNA ends (RACE) technique. The AOXs gene family members in P. persalinus was systematically identified by screening the genome using the conserved Pfam domain PF01786. Bioinformatics approaches were applied to analyze the physicochemical properties, subcellular localization, gene structure, Scaffold localization, promoter cis-regulatory elements, and phylogenetic relationships of these genes. Expression analysis of P. persalinus AOXs gene family members were carried out by qRT-PCR during the trophic and cyst stages and under temperature stress. The result showed that the full-length cDNA of the AOX3 gene from P. persalinus was cloned, with a total length of 1 091 bp (GenBank No. PV953026), including a 47 bp 5'-UTR, a 150 bp 3'-UTR, and an ORF of 894 bp. Four AOXs gene family members (designated as PpAOX1~PpAOX4) were identified in the whole genome; All PpAOXs were predicted to be hydrophilic proteins without signal peptides and were primarily localized in the cytoplasm and mitochondria. Cis-acting regulatory element analysis of the promoter regions suggested that PpAOXs should be involved in responses to hypoxia, temperature stress, and defense-related stimuli. Phylogenetic analysis of 33 AOXs genes from 13 protist species revealed that the 4 AOXs members of P. persalinus clustered within a clade of Oligohymenophorean ciliates. qRT-PCR results showed that PpAOX1 was highly expressed during the trophic stage, whereas PpAOX2~PpAOX4 exhibited significant upregulation during the cyst stage. The PpAOXs gene family members displayed distinct expression patterns in response to temperature stress. This study provides critical insights for further investigating the biological functions of PpAOXs and deciphering the adaptive strategies of P. persalinus in response to environmental stress.

Key words： Pseudocohnilembus persalinus Alternative oxidase (AOX) Gene cloning Gene family Expression analysis

收稿日期: 2025-08-13

中图分类号： S943

基金资助:国家重点研发计划(2022YFD2401002); 海水养殖生物育种全国重点实验室自由探索课题(MBL25IR10); 中央引导地方科技发展专项(2023L3024)

通讯作者: ^*zhbio@fjnu.edu.cn;lnfeng@hotmail.com

引用本文:

林童, 曾红, 潘滢, 吕艳红, 林能锋. 水滴伪康纤虫交替氧化酶基因(AOX3)的克隆及其基因家族鉴定与表达分析[J]. 农业生物技术学报, 2026, 34(5): 1104-1117.
LIN Tong, ZENG Hong, PAN Ying, LYU Yan-Hong, LIN Neng-Feng. Cloning of the Alternative Oxidase Gene (AOX3) from Pseudocohnilembus persalinus and Identification and Expression Analysis of the AOX Gene Family. 农业生物技术学报, 2026, 34(5): 1104-1117.

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https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2026.05.015 或 https://journal05.magtech.org.cn/Jwk_ny/CN/Y2026/V34/I5/1104

[1] 崔龙波, 刘冉, 王琛, 等. 2015. 大菱鲆盾纤毛虫病的流行病学调查[J]. 科学养鱼, (3): 54-56.
(Cui L B, Liu R, Wang C, et al. 2015. Epidemiological investigation of scuticociliatosis in Scophthalmus maximus[J]. Scientific Fish Farming, (3): 54-56.)
[2] 杜佳垠. 2001. 海水养殖鱼类盾纤虫病[J]. 渔业现代化, (3): 12-13.
(Du J Y. Scuticociliatosis of mariculture fish[J]. 2001. Fishery Modernization, (3): 12-13.)
[3] 范鑫鹏,潘旭明. 2020. 中国盾纤亚纲和咽膜亚纲纤毛虫[M]. 科学出版社, 北京. pp. 84-86.
(Fan X P, Pan X M.2020. Scuticociliatia and Peniculia ciliates in China[M]. Science Press.. Beijing. pp. 84-86.)
[4] 雷韬, 袁澍, 刘文娟, 等. 2006. 交替氧化酶结构和功能研究进展[J]. 西北植物学报, 26(3): 649-654.
(Lei T, Yuan S, Liu W J, et al.2006. Research advances in the structure and function of alternative oxidase[J]. Acta Botanica Boreali-Occidentalia Sinica, 26(3): 649-654.)
[5] 林子钠, 孙佳佳, 范鑫鹏, 等. 2023. 基于电镜观察的三伪尖毛虫(Oxytricha trifallax)成包囊过程研究[J]. 电子显微学报, 42(2): 199-207.
(Lin Z N, Sun J J, Fan X P, et al.2023. Study on the encystment of Oxytricha trifallax (Ciliophora: Hypotrichia) based on electronmicroscopy observations[J]. Journal of Chinese Electron Microscopy Society, 42(2): 199-207.)
[6] 吕艳红, 曾红, 林童, 等. 2025. 水滴伪康纤虫PpCaMK1基因克隆及生物学特征分析[J].福建农业学报, 40(1): 29-37.
(Lv Y H, Zeng H, Lin T, et al.2025. Cloning and biological characteristics of PpCaMK1 in Pseudocohnilembus persalinus[J]. Fujian Journal of Agricultural Sciences, 40(1): 29-37.)
[7] 潘旭明, 马洪钢, 邵晨, 等. 2012. 海洋纤毛虫: 水滴伪康纤虫的口器发生及形态学重描述[J]. 水生生物学报, 36(3): 489-495.
(Pan X M, Ma H G, Shao C, et al.2012. Stomatogenesis and morphological redescription of Pseudocohnilembus persalinus (Ciliophora: Scuticociliatida)[J]. Acta Hydrobiologica Sinica, 36(3): 489-495.)
[8] 帅良, 殷菲胧, 廖玲燕, 等. 2021. 香蕉果实MaNPCI基因原核表达及多克隆抗体制备[J].南方农业学报, 52(7): 1753-1761.
(Shuai L, Yin F L, Liao L Y, et al.2021. Prokaryotic expression of banana MaNPC1 gene and preparation of its polyclonal antibody[J]. Journal of Southern Agriculture, 52(7): 1753-1761.)
[9] 宋茜, 帅良, 张赐楷, 等. 2024. 辣椒交替氧化酶全基因家族的鉴定及表达分析[J]. 核农学报, 38(3): 424-433.
(Song Q, Shuai L, Zhang C K, et al.2024. Identification and expression analysis of alternative oxidase gene family in pepper[J]. Journal of Nuclear Agricultural Sciences, 38(3): 424-433.)
[10] 宋微波, 赵元莙, 徐奎栋. 2003. 海水养殖中的危害性原生动物[M]. 科学出版社, 北京. pp. 2.
(Song W B, Zhao Y J, Xu K D.2003. Harmful Protozoa in Marine Aquaculture[M]. Science Press, Beijing, China. pp. 2.)
[11] 王慧, 马艺文, 乔正浩, 等. 2020. AOX基因家族的结构和功能特征分析[J]. 生物技术通报, 38(7): 160-170.
(Wang H, Ma Y W, Qiao Z H, et al.2020. Structural and functional characterization of AOX gene family[J]. Biotechnology Bulletin, 38(7): 160-170.)
[12] 徐飞. 2018. 交替氧化酶抑制剂研究进展及应用前景[J]. 生命科学, 30(6): 621-627.
(Xu F.2018. Research progress and application prospects of alternative oxidase inhibitors[J]. Chinese Bulletin of Life Sciences, 30(6): 621-627.)
[13] 许金慧, 赵新宇, 曲同飞, 等. 2024. 浒苔交替氧化酶(alternative oxidase)基因克隆与环境应答[J]. 海洋科学, 48(10): 1-12.
(Xu J H, Zhao X Y, Qu T F, et al.2024. Cloning and environmental response of the alternative oxidase gene in Ulva prolifera[J]. Marine Sciences, 48(10): 1-12.)
[14] 徐奎栋, 宋微波. 1999. 海洋贝类几种危害性纤毛虫的研究[J]. 中国水产科学, 6(2): 41-45.
(Xu K D, Song W B.1999. Study on some pathogenetic ciliates from the mantle cavity of marine molluscs[J]. Journal of Fishery Sciences of China, 6(2): 41-45.)
[15] 曾红, 林能锋, 吕艳红, 等. 2024. 一种水滴伪康纤虫基因表达分析的内参基因组合及应用[P]. 中国, 202310809843.5.
(Zeng H, Lin N F, Lv Y H, et al.2024. Internal reference gene combination and application for gene expression analysis of Pseudocohnilembus persalinus[P]. China, 202310809843.5.)
[16] 张琦, 王磊, 谷延华, 等. 2024. 普通菜豆(Phaseolus vulgaris L.)AOX基因家族鉴定与表达分析[J]. 黑龙江八一农垦大学学报, 36(2): 1-8.
(Zhang Q,Wang L,Gu Y H, et al.2024. Genome-wide identification and expression analysis of AOX gene family members in common bean(Phaseolus vulgaris)[J]. Journal of Heilongjiang Bayi Agricultural University, 36(2): 1-8.)
[17] Anastasios D T, Karleigh A H, Catherine R E, et al.2018. The human gut colonizer Blastocystis respires using complex Ⅱ and alternative oxidase to buffer transient oxygen fluctuations in the gut[J]. Frontiers in Cellular and Infection Microbiology, 8: 371-382.
[18] Buchmann K.2015. Impact and control of protozoan parasites in maricultured fishes[J]. Parasitology, 142(1): 168-177.
[19] Castro J A, Ferreira M D G, Silva R J S, et al.2017. Alternative oxidase (AOX) constitutes a small family of proteins in Citrus clementina and Citrus sinensis L. Osb[J]. PLOS ONE, 12(5): e0176878.
[20] Chaudhuri M, Ajayi W, Hill G C.1998. Biochemical and molecular properties of the Trypanosoma brucei alternative oxidase[J]. Molecular and Biochemical Parasitology, 95(1): 53-68.
[21] Chaudhuri M, Ott R D, Hill G C.2006. Trypanosome alternative oxidase: from molecule to function[J]. Trends in Parasitology, 22(10): 484-491.
[22] Chen C, Chen H, Zhang Y, et al.2020. TBtools: an integrative toolkit developed for interactive analyses of big biological data[J]. Molecular Plant, 13(8): 1194-1202.
[23] Cisneros D, Cuteto-Díaz E J, Medina-Gil T, et al.2022. Imidazoline-and benzamidine-based trypanosome alternative oxidase inhibitors: synthesis and structure-activity relationship studies[J]. ACS Medicinal Chemistry Letters, 13(2): 312-318.
[24] Considine M J, Holtzapffel R C, Day D A, et al.2002. Molecular distinction between alternative oxidase from monocots and dicots[J]. Plant Physiology, 129: 949-953.
[25] Craig W R, Fiona R, Fiona L H, et al.2004. Evidence for mitochondrial-derived alternative oxidase in the apicomplexan parasite Cryptosporidium parvum: A potential anti-microbial agent target[J]. International Journal for Parasitology, 34(3): 297-308.
[26] Deng S, Beatty W, Sibley L D.2025. Assessing the function of the alternative electron transport chain in the Cryptosporidium parvum mitosome[J]. mBio, 16(12): e0112025.
[27] Ding C Q, Ng S, Wang L, et al.2018. Genome-wide identification and characterization of alternative oxidase genes and their response under abiotic stresses in Camellia sinensis (L.) O. Kuntze[J]. Planta, 248(5): 1231-1247.
[28] Ebiloma G U, Balogun E O, Cuteto-Díaz E J, et al.2019. Alternative oxidase inhibitors: Mitochondrion-targeting as a strategy for new drugs against pathogenic parasites and fungi[J]. Medicinal Research Reviews, 39(5): 1553-1602.
[29] Folgueira I, Lamas J, Sueiro R A, et al.2020. Molecular characterization and gene expression modulation of the alternative oxidase in a scuticociliate parasite by hypoxia and mitochondrial respiration inhibitors[J]. Scientific Reports, 10(1): 11880.
[30] Harikrishnan R, Balasundaram C, Heo M S.2010. Scuticociliatosis and its recent prophylactic measures in aquaculture with special reference to South Korea Taxonomy, diversity and diagnosis of scuticociliatosis: Part Ⅰ Control strategies of scuticociliatosis: Part Ⅱ[J]. Fish & Shellfish Immunology, 29(1): 15-31.
[31] Henriquez F L, Mcbride J, Campbell S J, et al.2009. Acanthamoeba alternative oxidase genes: Identification, characterisation and potential as antimicrobial targets[J]. International Journal for Parasitology, 39(13): 1417-1424.
[32] Iglesias R, Paramá A, Álvarez M F, et al.2001. Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain)[J]. Diseases of Aquatic Organisms, 46(1): 47-55.
[33] Li J, Yang S, Wu Y, et al.2024. Alternative oxidase: from molecule and function to future inhibitors[J]. ACS Omega, 9: 12478-12499.
[34] Li Y, Wang Y, Zhang S, et al.2022. How ciliated protists survive by cysts: Some key points during encystment and excystment[J]. Frontiers in Microbiology, 13: 785502.
[35] Liu B, Zhang Z, Peng J, et al.2024. Exploring evolutionary pathways and abiotic stress responses through genome-wide identification and analysis of the alternative oxidase (AOX) gene family in common oat (Avena sativa)[J]. International Journal of Molecular Sciences, 25(17): 9383.
[36] Liu Y, Niu J, Ye F, et al.2024. Dynamic DNA N (6)-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryote Pseudocohnilembus persalinus[J]. Genome Research, 34(2): 256-271.
[37] Mallo N, Lamas J, Leiro J M.2013. Evidence of an alternative oxidase pathway for mitochondrial respiration in the scuticociliate Philasterides dicentrarchi[J]. Protist, 164(6): 824-836.
[38] Mallo N, Lamas J, Leiro J M.2014. Alternative oxidase inhibitors as antiparasitic agents against scuticociliatosis[J]. Parasitology, 141(10): 1311-1321.
[39] McDonald A E, Gospodaryov D V.2019. Alternative NAD (P)H dehydrogenase and alternative oxidase: Proposed physiological roles in animals[J]. Mitochondrion, 45: 7-17.
[40] Millar A H, Whelan J, Soole K L, et al.2011. Organization and regulation of mitochondrial respiration in plants[J]. Annual Review Plant Biology, 62: 79-104.
[41] Mogi T, Kita K.2010. Diversity in mitochondrial metabolic pathways in parasitic protists Plasmodium and Cryptosporidium[J]. Parasitology International, 59(3): 305-312.
[42] Nakamura K, Fujioka S, Fukumoto S, et al.2010. Trypanosome alternative oxidase, a potential therapeutic target for sleeping sickness, is conserved among Trypanosoma brucei subspecies[J]. Parasitology International, 59(4): 560-564.
[43] Paramá A, Castro R, Arranz J A, et al.2007. Scuticociliate cysteine proteinases modulate turbot leucocyte functions[J]. Fish & Shellfish Immunology, 23(5): 945-956.
[44] Paramá A, Castro R, Lamas J, et al.2007. Scuticociliate proteinases may modulate turbot immune response by inducing apoptosis in pronephric leucocytes[J]. International Journal of Parasitology, 37(1): 87-95.
[45] Pedra-Rezende Y, Bombaça A C S, Menna-Barreto R F S.2022. Is the mitochondrion a promising drug target in trypanosomatids?[J]. Memórias do Instituto Oswaldo Cruz, 117: e210379.
[46] Piazzon C, Lamas J, Castro R, et al.2008. Antigenic and cross-protection studies on two turbot scuticociliate isolates[J]. Fish & Shellfish Immunology, 25(4): 417-424.
[47] Piazzon C, Lamas J, Leiro J M.2011. Role of scuticociliate proteinases in infection success in turbot, Psetta maxima (L.)[J]. Parasite Immunology, 33(10): 535-544.
[48] Rogov A G, Sukhanova E I, Uralskaya L A, et al.2015. Alternative oxidase: Distribution, induction, properties, structure, regulation, and functions[J]. Biochemistry (Moscow), 79(13): 1615-1634.
[49] Sendra K M, Watson A K, Kozhevnikova E, et al.2022. Inhibition of mitosomal alternative oxidase causes lifecycle arrest of early-stage Trachipleistophora hominis meronts during intracellular infection of mammalian cells[J]. PLOS Pathogens, 18(12): e1011024.
[50] Suzuki T, Hashimoto T, Yabu Y, et al.2005. Alternative oxidase (AOX) genes of African trypanosomes: Phylogeny and evolution of AOX and plastid terminal oxidase families[J]. The Journal of Eukaryotic Microbiology, 52(4): 374-381.
[51] Suzuki T, Nihei C, Yabu Y, et al.2004. Molecular cloning and characterization of Trypanosoma vivax alternative oxidase (AOX) gene, a target of the trypanocide ascofuranone[J]. Parasitology International, 53: 235-245.
[52] Szal B, Rychter A M.2016. Alternative oxidase-never ending story[J]. Postępy Biochemii, 62(2): 138-148.
[53] Tomoo S, Yasutoshi K, Kimitoshi S, et al.2013. Structure of the trypanosome cyanide-insensitive alternative oxidase[J]. Proceedings of the National Academy of Sciences of the United States of America, 110(12): 4580-4585.
[54] Vanlerberghe G C.2013. Alternative oxidase: A mitochondrial respiratory pathway to maintain metabolic and signaling homeostasis during abiotic and biotic stress in plants[J]. International Journal of Molecular Sciences, 14(4): 6805-6847.
[55] Weaver R J, McDonald A E.2023. Mitochondrial alternative oxidase across the tree of life: Presence, absence, and putative cases of lateral gene transfer[J]. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1864(4): 149003.
[56] Xiong J,Wang G Y,Cheng J,et al.2015. Genome of the facultative scuticociliatosis pathogen Pseudocohnilembus persalinus provides insight into its virulence through horizontal gene transfer[J]. Scientific Reports, 5: 15470.
[57] Zhang A, Brew-Appiah R A T, York Z B, et al.2018. Genome-wide identification and analysis of the alternative oxidase gene family in diploid and hexaploid wheat[J]. PLOS ONE, 13(8): e0201439.
[58] Zhang H, Luo Y, Wang Y, et al.2024. Genome-wide identification and characterization of alternative oxidase (AOX) genes in foxtail millet (Setaria italica): Insights into their abiotic stress response[J]. Plants, 13(18): 2565.