• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

水滴伪康纤虫PpCaMK1基因克隆及生物学特征分析

Cloning and Biological Characteristics Analysis of PpCaMK1, a Calmodulin-Dependent Protein Kinase in Pseudocohnilembus persalinus

  • 摘要:
    目的 钙调素依赖蛋白激酶(Calmodulin-Dependent Protein Kinases,CaMKs)是钙离子介导的信号转导通路中的关键组成部分,可能成为治疗盾纤虫病的药物靶点。本研究试图克隆水滴伪康纤虫(Pseudocohnilembus persalinus)的钙调素依赖蛋白激酶基因,分析其蛋白结构、功能,并基于该蛋白的氨基酸序列构建相关纤毛虫的系统进化树,以阐明水滴伪康纤虫CaMK蛋白的进化特征及与相关纤毛虫的系统发育关系。
    方法 参考水滴伪康纤虫RNA-seq测序结果,利用cDNA末端快速克隆(RACE)技术获得水滴伪康纤虫钙调素依赖蛋白激酶基因的全长cDNA序列,将其命名为PpCaMK1;利用生物信息学方法对该基因的序列特征、基因结构进行分析,预测该基因编码蛋白的理化性质、结构域及蛋白结构等,并构建其与相关纤毛虫的系统发育树。
    结果 PpCaMK1基因包含有9个外显子和8个内含子,其cDNA全长为1 737 bp(GenBank序列号:PQ278249),其5′-UTR长度为56 bp,3′-UTR长度为307 bp,ORF总长度为1 374 bp,编码457个氨基酸残基。PpCaMK1总亲水性平均值为-0.802,不稳定指数为54.78,为亲水性不稳定蛋白。亚细胞定位预测分析表明PpCaMK1存在于在细胞质-细胞核中。该蛋白无跨膜结构域,无信号肽。蛋白质的二级结构分析结果显示该蛋白含有的无规卷曲占比达50.33%。构建的系统发育树显示,水滴伪康纤虫PpCaMK1与寡膜纲纤毛虫同源蛋白具有较近的亲缘关系。
    结论 本研究克隆了水滴伪康纤虫PpCaMK1基因的全长cDNA,阐明了该基因的结构,并初步分析了PpCaMK1蛋白的基本理化性质及结构特征,结果可作为进一步研究PpCaMK1功能的基础,并为深入探究水滴伪康纤虫钙调素依赖蛋白激酶基因家族提供参考资料。

     

    Abstract:
    Objective Calmodulin-dependent protein kinases (CaMKs) are key components of calcium ion-mediated signaling pathways and may serve as potential drug targets for the treatment of scuticociliatosis. In this study, we attempted to clone the CaMK gene from Pseudocohnilembus persalinus, a scuticociliate pathogen that infects certain maricultured animals, analyze the structure and function of the CaMK protein, and constructed a phylogenetic tree based on its amino acid sequence. In order to elucidate the evolutionary characteristics of CaMK proteins in P. persalinus and provide insights into the phylogenetic relationships among related ciliates.
    Method Based on the RNA-seq sequencing results of P. persalinus, the Rapid Amplification of cDNA Ends (RACE) technique was employed to obtain the full-length cDNA sequence of the CaMK gene,which was designated as PpCaMK1. Bioinformatics methods were subsequently employed to analyze its sequence features, gene structure, physicochemical properties, protein domains, protein structure, and phylogenetic relationships.
    Result The PpCaMK1 gene contains 9 exons and 8 introns. The full cDNA length is 1737 bp (GenBank accession number: PQ278249), including a 5'-UTR of 56 bp, a 3'-UTR of 307 bp, and an ORF of 1374 bp encoding 457 amino acids residues. The overall hydrophilicity average of PpCaMK1 is -0.802, with an instability index of 54.78, indicating that it was a hydrophilic and unstable protein. Subcellular localization predictions indicated that PpCaMK1 was distributed within the cytoplasm and nucleus. Notably, the protein lacks transmembrane domains and signal peptides. Analysis of the secondary structure composition revealed a significant proportion (50.33%) of random coils. Phylogenetic analysis showed that P. persalinus was closely related to ciliates in the class Oligohymenophorea based on PpCaMK1.
    Conclusion In this study, the full-length cDNA of the PpCaMK1 gene was successfully cloned, and its structure was elucidated. A preliminary analysis of the basic physicochemical properties and structural characteristics of the PpCaMK1 protein was also conducted. These findings provide a foundation for further functional studies of PpCaMK1 and offer valuable reference data for investigating the CaMK gene family in P. persalinus.

     

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