Cloning of Phoenixin-20 gene in Lateolabrax maculatus and the effects of starvation and re-feeding on its expression
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摘要: 【目的】明确花鲈Phoenixin-20基因(LmPNX-20)是否参与花鲈的摄食调控过程,进而揭示PNX-20在鱼类摄食调节机制中的作用,同时为丰富和完善花鲈内分泌调控网络提供理论依据。【方法】通过PCR和RACE克隆LmPNX-20基因序列,采用ExPASy、SignalP 5.0、ProtScale、NetPhos 3.1及ClustalX等在线软件进行生物信息学分析,并以实时荧光定量PCR检测分析LmPNX-20基因在花鲈各组织中的表达特征及不同饥饿复投喂策略[正常投喂(F)、饥饿3 d(H3)、饥饿3 d复投喂1 d(R3-1)、饥饿3 d复投喂2 d(R3-2)、饥饿3 d复投喂3 d(R3-3),以及饥饿7 d(H7)、饥饿7 d复投喂1d(R7-1)、饥饿7d复投喂2d(R7-2)、饥饿7d复投喂3d(R7-3)]对LmPNX-20基因表达的影响。【结果】LmPNX-20基因开放阅读框(ORF)长度为210 bp,共编码69个氨基酸残基;其编码蛋白分子量为7.87 kD,理论等电点(pI)为9.85。LmPNX-20氨基酸序列含有5个磷酸化位点,但不存在信号肽;LmPNX-20氨基酸序列与硬骨鱼类的PNX-20氨基酸序列相似性较高,为69.70%~89.86%。LmPNX-20基因在花鲈脑组织中呈广泛表达分布,尤其在垂体和下丘脑中的相对表达量较高。经饥饿复投喂处理后花鲈幼鱼全脑组织中的LmPNX-20基因表达发生明显变化,在经历3和7 d饥饿复投喂1 d的花鲈幼鱼全脑组织中LmPNX-20基因的相对表达量显著上调(P<0.05);且与饥饿3 d复投喂的花鲈幼鱼相比,饥饿7 d复投喂的花鲈幼鱼全脑组织LmPNX-20基因表达均呈上调趋势。【结论】LmPNX-20基因在鱼类的进化过程中较保守,且在生长轴(下丘脑、垂体和肝脏)和生殖轴(下丘脑、垂体和性腺)有较高表达水平,可能参与花鲈的生长及生殖等生理过程。不同饥饿复投喂处理影响花鲈幼鱼全脑组织LmPNX-20基因的表达,复投喂后该基因表达量明显升高,说明LmPNX-20基因对花鲈幼鱼摄食调控具有一定促进作用。
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关键词:
- 花鲈 /
- LmPNX-20基因 /
- 饥饿复投喂 /
- 摄食调节
Abstract: 【Objective】To determine whether the Phoenixin-20 gene(LmPNX-20) was involved in the regulation of the feeding mechanism of Lateolabrax maculatus, and to reveal the role of PNX-20 in the regulation mechanism of fish feeding, and to provide a theoretical basis for enriching and improving the endocrine regulatory network of L. maculatus.【Method】The LmPNX-20 gene sequence was cloned by PCR and RACE, and bioinformatics analysis was performed by online softwares ExPASy, SignalP 5.0, ProtScale, NetPhos 3.1 and ClustalX. The expression characteristics of LmPNX-20 gene in various tissues were analyzed by real-time fluorescence quantitative PCR detection and effects of different starvation and re-feeding strategies on the expression of LmPNX-20 gene were detected[normal feeding(F), starvation for 3 d(H3), starvation for and 3 d re-feeding for 1 d(R3-1), starvation for 3 d and re-feeding for 2 d(R3-2), starvation for and 3 d re-feeding for 3 d(R3-3), starvation for 7 d(H7), starvation for 7 d and re-feeding for 1 d(R7-1), starvation for 7 d and re-feeding for 2 d(R7-2), starvation for 7 d and re-feeding for 3 d(R7-3)]. 【Result】LmPNX-20 gene had open reading frame(ORF) of 210 bp, encoding 69 amino acids residues. The encoded molecular weight of LmPNX-20 protein was 7.87 kD, with theoretical isoelectric point(pI) of 9.85. LmPNX-20 amino acid sequence contained 5 phosphorylation sites, but there was no signal peptide. Homology analysis of the amino acid sequence revealed that LmPNX-20 shared a high similarity of 69.70% to 89.86% with PNX-20 sequences from other bony fish species. LmPNX-20 gene was widely distributed in the brain tissue, with higher expression levels observed in the hypothalamus and pituitary. After starvation fasting and re-feeding treatments, the expression of LmPNX-20 gene in the whole brain tissue of juvenile L. maculatus showed obvious changes. After 3 and 7 d of starvation and re-feeding for 1 d, there was significant up-regulation of LmPNX-20 gene expression(P<0.05). After starvation for 7 d and re-feeding, the expression level of LmPNX-20 was upregulated compared to that after 3 d of starvation and re-feeding. 【Conclusion】LmPNX-20 gene is relatively conserved along fish evolution. Moreover, the expression level is high in the growth axis(hypothalamus, pituitary and liver) and reproductive axis(hypothalamus, pituitary and gonad), which may be involved in the physiological process of growth and reproduction of L. maculatus. Different starvation and re-feeding treatments affect the expression of LmPNX-20 gene in the whole brain tissue of juvenile L. maculatus, and the expression of this gene is greatly increased after re-feeding, that is, LmPNX-20 gene has a certain promotion effect on the feeding regulation of juvenile L. maculatus. -
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胡玲珠,周宣羽,张顺,徐善良.2021.饥饿再投喂对花鲈幼鱼生理生化指标的影响[J].宁波大学学报(理工版),34(4):8-14.[Hu L Z,Zhou X Y,Zhang S,Xu S L.2021.Effects of starvation and refeeding on physiological and biochemical indices in juvenile of Lateolabrax japonicus[J].Journal of Ningbo University(Natural Science&Engineering Edition),34(4):8-14.]doi: 10.3969/j.issn.1001-5132.2021.04.002 李嘉悦,刘立,何玲玲,侯悦晨.2022.神经肽Phoenixin的表达分布与功能机制的研究进展[J].中国实用神经疾病杂志,25(5):633-637.[Li J Y,Liu L,He L L,Hou Y C.2022.Research progress on the expression distribution and functional mechanism of neuropeptide Phoenix[J].Chinese Journal of Practical Neurological Diseases,25(5):633-637.]doi: 10.12083/SYSJ.220048. 田娟,何艮,麦康森,周慧慧.2016.鱼类食欲调控研究进展[J].动物营养学报,28(4):984-998.[Tian J,He G,Mai K S,Zhou H H.2016.Appetite regulation in fishes[J].Chinese Journal of Animal Nutrition,28(4):984-998.]doi: 10.3969/j.issn.1006-267x.2016.04.004. 王妹.2019.Phoenixin在金钱鱼生长和生殖中的作用研究[D].湛江:广东海洋大学.[Wang M.2019.Study on the role of Phoenixin in the growth and reproduction of spot-ted scat,Scatophagus argus[D].Zhanjiang:Guangdong Ocean University.]doi: 10.27788/d.cnki.ggdhy.2019.000090. 温海深,张美昭,李吉方,何峰,李昀.2016.我国花鲈养殖产业现状与种子工程研究进展[J].渔业信息与战略,31(2):105-111.[Wen H S,Zhang M Z,Li J F,He F,Li Y.2016.Research progress of aquaculture industry and its seed engineering in spotted sea bass (Lateolabrax macula-tus) of China[J].Modern Fisheries Information,31(2):105-111.]doi: 10.13233/j.cnki.fishis.2016.02.005. Álvarez A,García García B,Garrido M D,Hernández M D.2008.The influence of starvation time prior to slaughter on the quality of commercial-sized gilthead seabream (Sparus aurata) during ice storage[J].Aquaculture,284(1-4):106-114.doi: 10.1016/J.AQUACULTURE.2008.07.025.
Billert M,Rak A,Nowak K W,Skrzypski M.2020.Phoenixin:More than reproductive peptide[J].International Journal of Molecular Sciences,21(21):8378.doi: 10.3390/ijms21218378.
Clarke S A,Dhillo W S.2019.Phoenixin and its role in repro-ductive hormone release[J].Seminars in Reproductive Medicine,37(4):191-196.doi: 10.1055/s-0039-3400964.
Feng K,Luo H R,Hou M X,Li Y M,Chen J,Zhu Z Y,Hu W.2018.Alternative splicing of GnRH2 and GnRH2-associated peptide plays roles in gonadal differentiation of the rice field eel,Monopterus albus[J].General and Comparative Endocrinology,267:9-17.doi: 10.1016/j.ygcen.2018.05.021.
Gasparini S,Stein L M,Loewen S P,Haddock C J,Soo J,Fer-guson A V,Kolar G R,Yosten G L C,Samson W K.2018.Novel regulator of vasopressin secretion:Phoenixin[J].American Journal of Physiology.Regulatory,Integrative and Comparative Physiology,314(4):R623-R628.doi: 10.1152/ajpregu.00426.2017.
Goebel-Stengel M,Wang L X.2013.Central and peripheral expression and distribution of NUCB2/nesfatin-1[J].Cur-rent Pharmaceutical Design,19(39):6935-6940.doi: 10.2174/138161281939131127124814.
Grover H M,Smith P M,Ferguson A V.2020.Phoenixin influences the excitability of nucleus of the solitary tract neu-rones,effects which are modified by environmental and glucocorticoid stress[J].Journal of Neuroendocrinology,32(6):e12855.doi: 10.1111/jne.12855.
Guvenc G,Altinbas B,Kasikci E,Ozyurt E,Bas A,Udum D,Niaz N,Yalcin M.2019.Contingent role of phoenixin and nesfatin-1 on secretions of the male reproductive hormones[J].Andrologia,51(11):e13410.doi: 10.1111/and.13410.
Jiang J H,He Z,Peng Y L,Jin W D,Mu J,Xue H X,Wang Z,Chang M,Wang R.2015a.Effects of phoenixin-14 on anxiolytic-like behavior in mice[J].Behavioural Brain Research,286:39-48.doi: 10.1016/j.bbr.2015.02.011.
Jiang J H,He Z,Peng Y L,Jin W D,Wang Z,Mu L Y,Chang M,Wang R.2015b.Phoenixin-14 enhances memory and mitigates memory impairment induced by Aβ1-42 and sco-polamine in mice[J].Brain Research,1629:298-308.doi: 10.1016/j.brainres.2015.10.030.
Lyu R M,Cowan A,Zhang Y,Chen Y H,Dun S L,Chang J K,Dun N J,Luo J J.2018.Phoenixin:A novel brain-gut-skin peptide with multiple bioactivity[J].Acta Pharmacologica Sinica,39:770-773.doi: 10.1038/aps.2017.195.
Lyu R M,Huang X F,Zhang Y,Dun S L,Luo J J,Chang J K,Dun N J.2013.Phoenixin:A novel peptide in rodent sen-sory ganglia[J].Neuroscience,250:622-631.doi: 10.1016/j.neuroscience.2013.07.057.
Mcilwraith E K,Belsham D D.2018.Phoenixin:Uncovering its receptor,signaling and functions[J].Acta Pharmacologica Sinica,39(5):774-778.doi: 10.1038/aps.2018.13.
Morton G J,Cummings D E,Baskin D G,Barsh G S,Schwartz M W.2006.Central nervous system control of food intake and body weight[J].Nature,443(7109):289-295.doi: 10.1038/nature05026.
Pałasz A,Janas-Kozik M,Borrow A,Arias-Carrión O,Worthing-ton J J.2018.The potential role of the novel hypothalamic neuropeptides nesfatin-1,phoenixin,spexin and kisspeptin in the pathogenesis of anxiety and anorexia nervosa[J].Neurochemistry International,113:120-136.doi: 10.1016/j.neuint.2017.12.006.
Pałasz A,Rojczyk E,Bogus K,Worthington J J,Wiaderkiewicz R.2015.The novel neuropeptide phoenixin is highly coexpressed with nesfatin-1 in the rat hypothalamus,an immunohistochemical study[J].Neuroscience Letters,592:17-21.doi: 10.1016/j.neulet.2015.02.060.
Pałasz A,Tyszkiewicz-Nwafor M,Suszka-Świtek A,Bacopou-lou F,Dmitrzak-Węglarz M,Dutkiewicz A,SłopieńA,Janas-Kozik M,Wilczyński K M,FilipczykŁ,Bogus K,Rojczyk E,Paszyńska E,Wiaderkiewicz R.2021.Longitu-dinal study on novel neuropeptides phoenixin,spexin and kisspeptin in adolescent inpatients with anorexia nervosaAssociation with psychiatric symptoms[J].Nutritional Neu-roscience,24(11):896-906.doi: 10.1080/1028415X.2019.1692494.
Prinz P,Scharner S,Friedrich T,Schalla M,Goebel-Stengel M,Rose M,Stengel A.2017.Central and peripheral expres-sion sites of phoenixin-14 immunoreactivity in rats[J].Biochemical and Biophysical Research Communications,493(1):195-201.doi: 10.1016/j.bbrc.2017.09.048.
Samson W K,Zhang J V,Avsian-Kretchmer O,Cui K,Yosten G L C,Klein C,Lyu R M,Wang Y X,Chen X Q,Yang J,Price C J,Hoyda T D,Ferguson A V,Yuan X B,Chang JK,Hsueh A J W.2008.Neuronostatin encoded by the somatostatin gene regulates neuronal,cardiovascular,and metabolic functions[J].Journal of Biological Chemistry,283(46):31949-31959.doi: 10.1074/jbc.M804784200.
Schalla M A,Stengel A.2018.The role of ghrelin in anorexia nervosa[J].International Journal of Molecular Sciences,19(7):2117.doi: 10.3390/ijms19072117.
Schalla M,Prinz P,Friedrich T,Scharner S,Kobelt P,GoebelStengel M,Rose M,Stengel A.2017.Phoenixin-14injected intracerebroventricularly but not intraperitoneally stimulates food intake in rats[J].Peptides,96:53-60.doi: 10.1016/j.peptides.2017.08.004.
Shimizu H,Ohi S,Okada S,Mori M.2009.Nesfatin-1:An overview and future clinical application[J].Endocrine Jour-nal,56(4):537-543.doi: 10.1507/ENDOCRJ.K09E-117.
Stein L M,Tullock C W,Mathews S K,Garcia-Galiano D,Elias CF,Samson W K,Yosten G L C.2016.Hypotha-lamic action of phoenixin to control reproductive hormone secretion in females:Importance of the orphan G proteincoupled receptor Gpr173[J].American Journal of Physiology.Regulatory,Integrative and Comparative Physiology,311(3):R489-R496.doi: 10.1152/ajpregu.00191.2016.
Suszka-Świtek A,Pałasz A,FilipczykŁ,Menezes I C,Mordecka-Chamera K,Angelone T,Bogus K,Bacopoulou F,Worthington J J,Wiaderkiewicz R.2019.The GnRH ana-logues affect novel neuropeptide SMIM20/phoenixin and GPR173 receptor expressions in the female rat hypothalamic-pituitary-gonadal (HPG) axis[J].Clinical and Experimental Pharmacology and Physiology,46(4):350-359.doi: 10.1111/1440-1681.13061.
Treen A K,Luo V,Belsham D D.2016.Phoenixin activates immortalized GnRH and kisspeptin neurons through the novel receptor GPR173[J].Molecular Endocrinology,30(8):872-888.doi: 10.1210/me.2016-1039.
Ullah K,ur Rahman T,Wu D D,Lin X H,Liu Y,Guo X Y,Leung P C K,Zhang R J,Huang H F,Sheng J Z.2017.Phoenixin-14 concentrations are increased in association with luteinizing hormone and nesfatin-1 concentrations in women with polycystic ovary syndrome[J].Clinica Chi-mica Acta,471:243-247.doi: 10.1016/j.cca.2017.06.013.
Wang J,Zheng B,Yang S,Tang X Y,Wang J H,Wei D.2020.The protective effects of phoenixin-14 against lipopolysaccharide-induced inflammation and inflammasome acti-vation in astrocytes[J].Inflammation Research,69(8):779-787.doi: 10.1007/s00011-020-01355-9.
Wang M,Deng S P,Chen H P,Jiang D N,Tian C X,Yang W,Wu T L,Zhu C H,Zhang Y,Li G L.2018.Phoenixin par-ticipated in regulation of food intake and growth in spotted scat,Scatophagus argus[J].Comparative Biochemistry and Physiology.Part B:Biochemistry and Molecular Biology,226:36-44.doi: 10.1016/j.cbpb.2018.07.007.
Wernecke K,Lamprecht I,Jöhren O,Lehnert H,Schulz C.2014.Nesfatin-1 increases energy expenditure and reduces food intake in rats[J].Obesity,22(7):1662-1668.doi: 10.1002/oby.20736.
Yosten G L C,Lyu R M,Hsueh A J W,Avsian-Kretchmer O,Chang J K,Tullock C W,Dun S L,Dun N,Samson W K.2013.A novel reproductive peptide,phoenixin[J].Journal of Neuroendocrinology,25(2):206-215.doi: 10.1111/j.1365-2826.2012.02381.x.
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