Fang-1,一个参与血糖调节的新基因

A Novel Gene Involved in Blood Glucose Regulation

目的克隆血糖调节相关基因。方法按本室以往建立的方法制备大鼠血糖自动调节模型,以灌输生理盐水为对照;取其骨骼肌,采用差异显示技术(DDRT-PCR),获得差异表达基因片段,经狭缝杂交和Northern杂交分析,排除假阳性片段,确定真正的差异表达序列标签(EST),并以其为探针筛选大鼠骨骼肌cDNA文库;将新基因的全长编码区克隆到pEGFP,瞬时转染L-6TG细胞48h后,荧光显微镜观察蛋白质在细胞中定位。结果获得一个新的全长cDNA,命名为Fang-1,GenBank收录号为AF399873。Blast软件(NCBI)分析显示Fang-1是人类基因AK001644在大鼠中的同源物,其编码区核苷酸同源性为82%,表明该家族蛋白具有保守性。高浓度葡萄糖刺激大鼠后与对照大鼠相比,Fang-1的表达上调;Fang-1编码的蛋白质在细胞核和细胞质均存在。结论从大鼠骨骼肌中克隆到一个参与血糖调节的新基因,该基因编码蛋白在细胞质和细胞核均存在,并通过其表达上调控制或部分控制某些目前未知的机制而达到调节血糖水平的作用。

Objective To clonea a novel gene relatied to blood glucose regulation.Methods Constract rat model of autonomous regulation through jugular vein right atrium intubation of high concentration of glucose,the control rats were injected with0.9%NaCl and skeletal muscle was separated.The differentially expressed fragments were identified by differential display technology(DDRT-PCR).After slot blot and Northern blot analysis,we excluded the artificial positive fragments and got the true EST (expression sequence tag)differentially expressed.These positive EST were used as probes to screen cDNA library of rat skeletal muscle,the coding region of full-length gene was cloned to pEGFP and L-6TG cell line was cultured and transiently transfected with the lipofectamine reagent.After48h,intact cells were examined with fluorescence microscope.Results We got a novel full-length cDNA,named as Fang-1.GenBank Accession No.is AF399873.Using blast software(NCBI),we found Fang-1is rat homologue of human AK001644.They share82%identical nucleotides,indicating the family proteins are very conserved.After high concentration of glucose stimulation compared with control rats,the expression of Fang-1was up-regulated and the expression product of Fang-1was localized in both cytoplasm and cell nucleus.Conclusions A novel gene related to blood glucose regulation was cloned from rat skeletal muscle.The expression product of Fang-1was localized in both cytoplasm and cell nucleus.The gene can regulate blood glucose level by controlling some mechanisms unknown now with down-regulation expression.