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1 d(R7-1)、饥饿7 d复投喂2 d(R7-2)、饥饿7 d复投喂3 d(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.
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 star-vation 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 rea-ding 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 up-regulated 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 re-productive 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.
基金项目
国家重点研发计划项目(2022YFD2400503)
广东省基础与应用基础研究基金(2023A1515030022)
广州市科技计划项目(2023B03J1304)
中国水产科学研究院中央级公益性科研院所基本科研业务费专项(2022YFD2400503)
维普
万方数据