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

【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.