斑马鱼角蛋白8基因克隆、组织分布及时空表达分析

Cloning,tissue distribution,and spatiotemporal expression analysis of the Keratin 8 gene in Zebrafish(Danio rerio)

研究旨在分析斑马鱼(Danio rerio)角蛋白8(Keratin 8,Krt8)基因的分子特性,组织分布及病原菌感染后的动态表达模式。通过克隆斑马鱼Krt8基因CDS序列,进行生物信息学分析。利用实时荧光定量PCR技术,研究Krt8基因在健康斑马鱼肠道、肝脏、肾脏和皮肤组织中的表达分布,并分析了海藻希瓦氏菌(Shewanella algae)感染后,在各组织中的时空表达模式。研究结果显示,斑马鱼Krt8基因CDS区序列编码一个含520个氨基酸的蛋白质,其分子量为57.76 kDa,且具有14个磷酸化位点。对构建的系统发育进化树分析表明,斑马鱼Krt8的氨基酸序列与安水金线鲃(Sinocyclocheilus anshuiensis)的亲缘关系最近。此外,Krt8基因在斑马鱼肠道、肝脏、肾脏和皮肤组织中均有分布,其中在肝脏中的表达水平最高。海藻希瓦氏菌感染后,Krt8基因在斑马鱼检测组织中呈现差异表达,其中在肠道中表达下调,在肝脏和肾脏组织中呈现先升高再下降的趋势。综上所述,斑马鱼Krt8可能参与海藻希瓦氏菌感染引起的机体免疫应答或病理生理过程,为进一步研究Krt8蛋白的功能和作用机理提供参考资料。

To analyze the molecular characteristics,tissue distribution,and dynamic expression patterns post-pathogen infection of Keratin 8(Krt8)gene in zebrafish(Danio rerio),this study cloned the coding sequence(CDS)of the zebrafish Krt8 and conducted a bioinformatics analysis.The expression distribution of the Krt8 gene in the intestine,liver,kidney,and skin tissues of healthy zebrafish was examined using real-time quantitative PCR,and its spatiotemporal expression pattern following infection with Shewanella algae was analyzed.The results showed that the CDS of the zebrafish Krt8 gene encoded a protein consisting of 520 amino acids with a molecular weight of 57.76 kDa,featuring 14 phosphorylation sites.Phylogenetic tree analysis indicated a close evolutionary relationship between the amino acid sequence of zebrafish Krt8 and that of Sinocyclocheilus anshuiensis.Furthermore,the Krt8 gene was expressed in the intestine,liver,kidney,and skin tissues of zebrafish,with the highest expression level observed in the liver.Following infection with S.algae,differential expression patterns of Krt8 were noted in the tested tissues,with a downregulation in the intestine and an initial increase followed by a decrease in the liver and kidney.In summary,the findings of this study suggest that Krt8 in zebrafish may play a role in the organism's immune response or pathophysiological processes induced by S.algae infection.These insights provide a valuable reference for further investigations into the functional mechanisms and roles of Krt8 protein.