Cloning of the SMIT1 and SMIT2 Genes in Gymnocypris przewalskii and Its Response to Alkaline Environment
[Objective]The sodium/myo-inositol cotransporter(SMIT)in mammalian is a solute carrier(SLC)superfamily protein that has two homologs:SMIT1 and SMIT2.The experiment was aimed to investigate the response of SMIT1 and SMIT2 genes in Gymnocypris przewalskii to alkaline environment,so as to lay the foundation for understanding the molecular mechanisms of SMIT1 and SMIT2 genes in myo-inositol transport under alkaline condition.[Method]The CDS region of SMIT1 and SMIT2 genes were amplified using PCR and cloned,followed by bioinformatics analysis.The expression of SMIT1 and SMIT2 gene in eight tissues,such as gill,kidney and brain,of Gymnocypris przewalskii in freshwater environment was detected using Real-time quantitative PCR.Additionally,the expression of the two genes in gill and kidney of Gymnocypris przewalskii under different alkaline stress(0,J25,J50,J75 and J100)conditions were investigated,and the changes in myo-inositol content were detected using gas chromatography-mass spectrometry(GC-MS)technology.[Result]The CDS sequence of SMIT1 gene was 2 130 bp,encoding a total of 709 amino acids,the amino acid sequence of SMIT1 gene showed the highest similarity of 86.5％with Sinocyclocheilus rhinocerous,indicating a closer phylogenetic relationship with Onychostoma macrolepis.The CDS sequence of SMIT2 gene was 2 016 bp,encoding a total of 671 amino acids,the amino acid sequence of SMIT2 gene showed the highest similarity of 90.3％with Onychostoma macrolepis,indicating a closer phylogenetic relationship with Sinocyclocheilus rhinocerous and Onychostoma macrolepis.SMIT1 and SMIT2 both belonged to the SLC5 family members of the solute superfamily,SMIT1 protein contained two conserved domains of the SLC family.Real-time quantitative PCR results indicated that both SMIT1 and SMIT2 genes were expressed in various tissues of Gymnocypris przewalskii in freshwater environment,the expression of SMIT1 gene was higher in brain and kidney,while the expression of SMIT2 gene was highest in kidney,both of which were significantly higher than that in other tissues(P＜0.05).With increasing alkalinity,compared with control group,the expression of SMIT1 gene in gill of Gymnocypris przewalskii at J25,J50 and J100 was significantly increased(P＜0.05),and the overall expression in kidney was significantly increased(P＜0.05).On the other hand,the expression of SMIT2 gene in gill of Gymnocypris przewalskii at J25 was significantly increased,and the expression in kidney at J50,J75 and J100 was significantly decreased(P＜0.05).The GC-MS results showed that the myo-inositol content in gill of Gymnocypris przewalskii was significant higher than that in control group(P＜0.05),but there was no significant change of the myo-inositol content in kidney with control group(P＞0.05).[Conclusion]The CDS length of SMIT1 and SMIT2 genes were 2 130 and 2 016 bp,encoding 709 and 671 amino acids.SMIT1 and SMIT2 genes were highly expressed in brain and kidney of Gymnocypris przewalskii,respectively.The expression patterns of SMIT1 and SMIT2 genes in gill and kidney of Gymnocypris przewalskii under different alkalinity stresses.The myo-inositol content in gill was significantly increased and reached its peak at J50,with no significant change in kidney.The results provided a reference basis for further exploring the molecular mechanism of Gymnocypris przewalskii adaptation to alkaline environment.
万方数据
维普
