青海湖裸鲤SMIT1和SMIT2基因克隆及其对碱度环境的应答

Cloning of the SMIT 1 and SMIT 2 Genes in Gymnocypris przewalskiiand Its Response to Alkaline Environment

【目的】哺乳动物钠/肌醇共转运蛋白(sodium/myo-inositol cotransporter, SMIT)有2个同源体,分别是SMIT1和SMIT2,属于溶质载体(SLC)超家族。试验旨在探究青海湖裸鲤SMIT1、SMIT2基因对碱度环境的应答,为后续SMIT1和SMIT2基因在碱度环境下参与肌醇转运的分子机制奠定基础。【方法】利用PCR技术扩增、克隆SMIT1、SMIT2基因CDS区,并对其进行生物信息学分析;利用实时荧光定量PCR技术检测SMIT1和SMIT2基因在淡水环境下青海湖裸鲤鳃、肾脏、脑等8个组织中的表达情况,以及不同碱度胁迫(0、J25、J50、J75、J100)下鳃和肾脏组织中2个基因表达的规律;利用气相色谱质谱法(GC-MS)检测不同碱度胁迫下鳃和肾脏组织中的肌醇含量变化。【结果】SMIT1基因CDS区为2 130 bp,共编码709个氨基酸,其氨基酸序列与犀角金线鲃相似性最高(86.5%),与多鳞白甲鱼亲缘关系最近;SMIT2基因CDS区为2 016 bp,共编码671个氨基酸,其氨基酸序列与多鳞白甲鱼的相似性最高(90.3%),与犀角金线鲃、多鳞白甲鱼的亲缘关系较近。SMIT1和SMIT2均属于溶质体超家族的SLC5家族成员,其中SMIT1蛋白包含2个SLC家族保守结构域。实时荧光定量PCR结果显示,淡水环境下SMIT1和SMIT2基因在青海湖裸鲤不同组织中均有表达,其中,SMIT1基因在脑和肾脏中表达量较高,SMIT2基因在肾脏中表达量最高,均显著高于其他组织(P<0.05)。随着碱度的升高,与对照组相比,J25、J50、J100时SMIT1基因在青海湖裸鲤鳃中表达量均显著升高(P<0.05),在肾脏中整体表达量均显著升高(P<0.05);J25时SMIT2基因在青海湖裸鲤鳃中表达量显著升高(P<0.05),J50、J75、J100时在肾脏中表达量均显著降低(P<0.05)。GC-MS结果显示,随着碱度的升高,青海湖裸鲤鳃中肌醇含量均显著高于对照组(P<0.05);肾脏中肌醇含量与对照组相比无显著变化(P>0.05)。【结论】SMIT1、SMIT2基因CDS区分别长2 130和2 016 bp,分别编码709和671个氨基酸,SMIT1基因在青海湖裸鲤脑组织中高表达,而SMIT2基因在青海湖裸鲤肾脏中高表达。不同碱度胁迫下SMIT1、SMIT2基因在青海湖裸鲤鳃、肾脏中的表达规律不同;肌醇含量在鳃组织中显著升高,且在J50时达到最高,在肾脏中变化不显著。研究结果为进一步探究青海湖裸鲤适应碱度环境的分子机制提供参考依据。

【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 SMIT 1 and SMIT 2 genes in Gymnocypris przewalskii to alkaline environment,so as to lay the foundation for understanding the molecular mechanisms of SMIT 1 and SMIT 2 genes in myo-inositol transport under alkaline condition.【Method】The CDS region of SMIT 1 and SMIT 2 genes were amplified using PCR and cloned,followed by bioinformatics analysis.The expression of SMIT 1 and SMIT 2 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 SMIT 1 gene was 2130 bp,encoding a total of 709 amino acids,the amino acid sequence of SMIT 1 gene showed the highest similarity of 86.5%with Sinocyclocheilus rhinocerous,indicating a closer phylogenetic relationship with Onychostoma macrolepis.The CDS sequence of SMIT 2 gene was 2016 bp,encoding a total of 671 amino acids,the amino acid sequence of SMIT 2 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 SMIT 1 and SMIT 2 genes were expressed in various tissues of Gymnocypris przewalskii in freshwater environment,the expression of SMIT 1 gene was higher in brain and kidney,while the expression of SMIT 2 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 SMIT 1 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 SMIT 2 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 SMIT 1 and SMIT 2 genes were 2130 and 2016 bp,encoding 709 and 671 amino acids.SMIT 1 and SMIT 2 genes were highly expressed in brain and kidney of Gymnocypris przewalskii,respectively.The expression patterns of SMIT 1 and SMIT 2 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.