银鲫果糖-1,6-二磷酸酶的分子克隆与表达分析

MOLECULAR CLONING AND EXPRESSION ANALYSIS OF FRUCTOSE-1,6-BISPHOSPHATASE IN GIBEL CARP

果糖-1,6-二磷酸酶(EC 3.1.3.11)是糖异生中的关键限速酶之一,在糖代谢中起重要作用。哺乳动物存在肝脏型和肌肉型两种果糖-1,6-二磷酸酶同工酶,分别由Fbp1和Fbp2编码。银鲫作为我国重要的经济养殖鱼类,尚无果糖-1,6-二磷酸酶基因的有关资料,其组织分布特征和胚胎发育模式亦不清楚。本研究采用RACE方法从银鲫原肠胚SMART cDNA文库中扩增了果糖-1,6-二磷酸酶基因的全长cDNA,其长度为1170 bp,编码337个氨基酸残基,多重序列比对和系统发育分析表明该基因为肝脏型果糖-1,6-二磷酶。RT-PCR分析虽在银鲫的肝、脑、心、脾、肾、肠、肌肉和卵巢组织中皆能检测到该基因的表达,但以肝组织的表达量最高。Western Blot检测表明,肝脏组织除有一条与其他组织(肌肉除外)共有的蛋白带之外,还有一条特异带;肌肉中有不同于其他组织的特异带。成熟卵子和不同发育阶段胚胎的RT-PCR和Western Blot分析都可检测到母源的CagFbp转录本和蛋白,且其转录本从原肠期开始上升,到神经胚时迅速上升到较高水平,其蛋白从尾芽期以后出现一条比母源蛋白分子量小、与肝脏的特异带大小基本相同的蛋白带。这些结果证实本研究克隆的CagFbp为肝脏型,且鱼类至少存在肝脏型和肌肉型两种果糖-1,6-二磷酸酶同工酶。

Fructose-1,6-bisphosphatase is one of the key rate-limiting enzymes in gluconeogenesis,which plays important roles in carbohydrate metabolism.Mammals have two isoforms,liver and muscle fructose-1,6-bisphosphatase encoded by Fbp1 and Fbp2 respectively.Gibel carp is widely cultured as an economic fish in China.However,the fructose-1,6-bisphosphatase gene has not been elucidated in teleosts,especially its tissue distribution in adult fish and spatiotemporal expression in embryogenesis.In this study,we cloned the full-length cDNA of gibel carp Fbp1 by RACE polymerase chain reaction from the gastrula embryo SMART cDNA library,and we also examined its expression pattern in the tissues of adult fish and the developmental process of embryos in this fish by gene specific primers.The full length sequence of CagFbp1 consists of 1170 base pairs which encodes 337 amino acids.Multiple alignment and phylogenetic analysis showed that the cloned gene was liver Fbp of gibel carp.The tissue expression pattern analysis by RT-PCR with specific primers showed that CagFbp1 was expressed in the liver,brain,heart,spleen,kidney,intestine,muscle and ovary,and the expression in the liver was obviously higher than others.At the same times,there were two protein bands in liver by western blot analysis,one was common in the detected tissue except muscle,and the other was specific for liver.However,only one band emerged in the muscle,which was specific for muscle tissues.Mature eggs and ontogenic analysis by RT-PCR and western blotting with specific primers showed that both the transcripts and proteins of CagFbp1 were maternal.The transcripts were increased from gastrula and reached a higher level in neurula till hatching.Interestingly,there was a new band with smaller molecular weight other than the maternal proteins after tail bud stage,which was similar to the liver specific band.These results indicated that the fructose-1,6-bisphosphatase gene cloned in gibel carp was liver isoform,and there might be at least two isoenzymes,the liver fructose-1,6-bisphosphatase and muscle one in teleost.