草鱼SGLT1/2基因克隆及葡萄糖处理对其mRNA表达的影响

作为重要的转运载体,SGLTs(钠依赖性葡萄糖协同转运蛋白,sodium-glucose cotransporters)在物质的吸收过程中发挥着关键的作用。SGLT1和SGLT2作为SGLTs家族的重要成员,参与调节体内的葡萄糖吸收,从而维持血糖的稳态。为研究SGLT1和SGLT2在草鱼葡萄糖吸收过程中的作用,本实验利用RT-PCR技术从草鱼前肠和肾脏中分别克隆了基因sglt1和sglt2,对其进行生物信息学分析,并利用荧光定量PCR技术检测其mRNA的表达。结果显示,草鱼sglt1和sglt2的开放阅读框(open reading frame,ORF)分别为1 977和1 989 bp,并分别编码658和662个氨基酸。经过预测,草鱼SGLT1和SGLT2蛋白均为14次跨膜结构。氨基酸序列比对及系统进化树分析结果显示,草鱼sglt1和sglt2与金鱼、金线鲃中sglt1和sglt2的亲缘关系最近。荧光定量PCR结果显示,sglt1在草鱼肠道和肾脏组织中表达量较高,在其他组织中表达量较低;sglt2在草鱼肾脏组织中表达量最高,在其他组织中表达量较低。葡萄糖灌喂实验结果显示,草鱼前肠中sglt1和肾脏中sglt2的表达量在灌喂1 h后显著增加。在离体实验中,葡萄糖处理CIK细胞能够显著增加sglt1和sglt2的表达量。本研究为完善SGLTs在调控鱼类血糖稳态方面具有的功能提供了理论依据。

Cloning of grass carp (Ctenopharyngodon idella) SGLT1/2 genes and effect of glucose on its mRNA expression

As the co-transporters, SGLTs play a pivotal role in the absorption of the substance. As the important member of SGLTs, SGLT1 and SGLT2 are involved in keeping blood glucose homeostasis by regulation of glucose uptake. To investigate the role of SGLT1 and SGLT2 in the glucose uptake of grass carp (Ctenopharyngodon idellus), a series of experiment was performed. In this study, the grass carp SGLT1 and SGLT2 were cloned in foregut and kidney by RT-PCR, and the sequences were analyzed by bioinformatics and the mRNA expression were detected by Real-time PCR. The results showed that the ORF of grass carp SGLT1 and SGLT2 were 1977 bp and 1989 bp, which encode 658 and 662 amino acids. Based on amino acids sequence, the structure of SGLT1 and SGLT2 protein were 14 transmembrane, and the results of sequence alignment and phylogenetic tree showed that the grass carp SGLT1 and SGLT2 gene were closest to that of goldfish and sinocyclocheilus. The Real-time PCR results indicated that the higher expression of SGLT1 was in the intestine and kidney, and the low expression of SGLT1 was in other tissues of grass carp. The higher expression of SGLT2 was in the kidney, and the low expression of SGLT2 was in other tissues of grass carp. In OGTT experiment of grass carp, the SGLT1 mRNA expression was significantly increased in grass carp foregut, and the SGLT2 mRNA expression was markedly increased in grass crap kidney by glucose treatment 1h. In in vitro experiment, the SGLT1 and SGLT2 mRNA expression were markedly increased in the CIK cells by glucose treatment. The results will provide the data base for consummating SGLTs functions and theoretical foundation for investigating the regulatory of glucose homeostasis in fish.