青海湖裸鲤颗粒蛋白基因克隆及其参与盐碱适应机制研究

Cloning of granulin gene in Gymnocypris przewalskii and its involvement in salinity-alkalinity adaptation mechanism

  • 摘要: 【目的】探究青海湖裸鲤颗粒蛋白A基因(GrnA)和颗粒蛋白B基因(GrnB)的生物学功能,为揭示颗粒蛋白(Grn)在青海湖裸鲤耐盐碱过程中的作用机制提供理论依据。【方法】以青海湖裸鲤为研究对象,通过RACE和PCR克隆青海湖裸鲤GrnA基因cDNA序列和GrnB基因编码区(CDS)序列,利用实时荧光定量PCR检测青海湖裸鲤GrnAGrnB基因在各组织中的表达情况及在盐碱耐受过程中的表达变化,并分析二者与成骨分化特异性转录因子基因(Runx2)和补体基因C8g间的相关性。【结果】青海湖裸鲤GrnA基因cDNA序列全长653bp,其开放阅读框(ORF)465bp,共编码159个氨基酸残基;青海湖裸鲤GrnB基因CDS序列长1560 bp,共编码519个氨基酸残基。青海湖裸鲤与银鲫的GrnA氨基酸序列相似性高达89.61%,基于GrnA氨基酸序列相似性构建的系统发育进化树显示青海湖裸鲤与鲤和鲫的亲缘关系较近;青海湖裸鲤与多鳞白甲鱼的GrnB氨基酸序列相似性高达89.69%,基于GrnB氨基酸序列相似性构建的系统发育进化树显示青海湖裸鲤与多鳞白甲鱼和虎皮鱼的亲缘关系较近。GrnAGrnB基因在青海湖裸鲤不同组织中均有表达,且以脾脏中的相对表达量最高,显著高于其他组织(P<0.05,下同)。青海湖裸鲤鳃组织中的GrnA基因对盐胁迫浓度增加的响应速度最快,其次是脾脏和肾脏;GrnB基因则表现为在肾脏中的响应速度最快,其次是脾脏和鳃组织,但在鳃组织中能持续响应。在碱胁迫下,青海湖裸鲤GrnA基因在3个组织中呈不规律性表达变化;GrnB基因表现为在鳃组织中的响应最强,其次是肾脏。在盐碱混合胁迫下,GrnA基因在鳃组织中的响应强于脾脏,在肾脏中也有响应;GrnB基因在鳃组织中最先响应盐碱混合胁迫,其次是肾脏和脾脏。在青海湖裸鲤鳃组织中,GrnAGrnB基因在盐胁迫或碱胁迫下与Runx2基因呈正相关;在脾脏中,GrnA基因与C8g基因在盐胁迫下呈极显著正相关(P<0.01),碱胁迫下GrnAGrnB基因与C8g基因呈正相关。【结论】青海湖裸鲤GrnAGrnB基因除了在脾脏中高表达外,在鳃组织和肾脏中也有相应表达,参与青海湖裸鲤的盐碱适应过程,且表现为GrnA基因对盐胁迫的响应更明显,而GrnB基因对碱胁迫的响应更明显。此外,青海湖裸鲤GrnAGrnB基因在鳃组织中可能是通过调控Runx2基因影响鳃弓的生物矿化过程,在脾脏中可能是通过激活免疫调节过程,共同协助青海湖裸鲤适应高盐碱环境。

     

    Abstract: 【Objective】To investigate the biological functions of granulin A gene(GrnA) and granulin B gene(GrnB) in Gymnocypris przewalskii, and to provide theoretical basis for revealing the mechanism of the role of granulin(Grn) in the salinity and alkalinity tolerance process of G. przewalskii. 【Method】G. przewalskii was as research object, cDNA sequence of GrnA gene and the coding region(CDS) sequence of GrnB gene were cloned by RACE and PCR, and real-time quantitative PCR was used to detect the expression of GrnA and GrnB genes in various tissues of G. przewalskii and the changes during salinity and alkalinity tolerance. Correlations among the two genes and osteogenic differentiation-specific transcription factor gene(Runx2) and complement gene C8g were also analyzed. 【Result】The cDNA sequence of GrnA gene of G. przewalskii was 653 bp in length, with an open reading frame(ORF) of 465 bp, encoding 159 amino acid residues; the CDS sequence of GrnB gene was 1560 bp in length, encoding 519 amino acid residues. The amino acid sequence similarity of GrnA between G. przewalskii and Silver prussian carp was as high as 89.61%, and the phylogenetic tree constructed on the basis of the GrnA amino acid sequence similarity showed that G. przewalskii was more closely related to carp and crucian carp; and the amino acid sequence similarity of GrnB between G. przewalskii and Scaphesthes macrolepis was as high as 89.69%, and the phylogenetic tree constructed on the basis of the similarity of GrnB amino acid sequences showed that G. przewalskii was closely related to S. macrolepis and Puntius tetrazona. GrnA and GrnB genes were expressed in different tissues of G. przewalskii, with the highest expression in spleen, which was significantly higher than that in other tissues(P<0.05, the same below). The GrnA gene in gill tissue of G. przewalskii showed the fastest response to salt stress, followed by spleen and kidney; while the GrnB gene showed the fastest response in kidney, followed by spleen and gill, but sustained response in gill tissue. Under alkalinity stress, the GrnA gene showed irregular expression changes in the three tissues; the GrnB gene showed the strongest response in gill tissue, followed by kidney. Under salinity-alkalinity mixed stress, GrnA gene responded stronger in gill tissue than in spleen, and also responded in kidney; GrnB gene responded first in gill tissue, followed by kidney and spleen. In gill tissue, GrnA and GrnB genes were positively correlated with Runx2 gene under salinity or alkalinity stress; in spleen, GrnA gene was extremely significantly positively correlated with C8g gene under salinity stress(P<0.01), and GrnA and GrnB genes were positively correlated with C8g gene under alkalinity stress. 【Conclusion】In addition to high expression in spleen, GrnA and GrnB genes are also expressed in gill and kidney, which are involved in saline and alkaline adaptation in G. przewalskii, and GrnA gene shows a more obvious response to salinity stress, while GrnB gene shows a more obvious response to alkalinity stress. It is more likely that the GrnA and GrnB genes affect the biomineralization process of gill arches through the regulation of Runx2 gene in gill tissue and activate immune regulation in spleen, which together assists the adaptation to high salinity and alkalinity environments of G. przewalskii.

     

/

返回文章
返回