翘嘴红鲌肝脏G6Pase催化亚基的克隆以及摄食和饲料中碳水化合物对其表达的影响

Molecular cloning of hepatic glucose-6-phosphatase catalytic subunit from Erythroculter ilishaeformis: response of its expression to refeeding and carbohydrate in diet

本文采用RT—PCR和RACE法分离、克隆了翘嘴红鲌G6Pase催化亚基基因全长cDNA，共1900bp[不含poly（A）]，包括49bp5’非翻译区，1068bp阅读框以及含Poly（A）信号AATAAA的778bp3’非翻译区[不包括Poly（A）]。阅读框共编码355个氨基酸，分子量为39．89ku。序列比对分析表明翘嘴红鲌G6Pase与斑马鱼G6Pase的相似性高达95％，与鼠、狗、人G6Pase的相似性为63％，和光滑爪蟾、金头鲷、河豚等G6Pase的相似性分别为69％、55％、76％，并具有G6Pase特有的3个保守基序。为了研究摄食以及饲料中碳水化合物对G6Pase的影响，使用实时定量RT—PCR分别测定了饲喂等能但不含碳水化合物和含23．98％碳水化合物的饲料的翘嘴红鲌肝脏G6Pase基因的表达水平，在使用上述饲料饲喂8周后，禁食48h，然后测定禁食和摄食后3、6、12、24hG6Pase mRNA的表达量，结果显示摄食后12h，两组G6Pase的表达明显增加，说明摄食影响G6Pase基因的表达，禁食和摄食后3～12h，含糖组G6Pase基因的表达量为无糖组的2～4倍，这表明碳水化合物可以诱导G6Pase mRNA的表达。

Liver glucose-6-phosphatase （ G6Pase, EC 3.1.3.9）, one of the key enzymes in the glycolysis and glyconeogenesis, plays a key role in blood glucose homeostasis by catalyzing the dephosphorylation of glucose-6-phosphate（Glu-6P） to glucose. RT-PCR and RACE（rapid amplification cDNA ends ） was used for the isolation of the full length cDNA of G6Pase gene from liver of Erythroculter ilishaeformis. The cDNA was 1913 bp containing the 49 bp 5＇-untranslated region, 778 bp 3＇-untranslated region and 1068 bp open reading frame, which encoded 355 amino acid with a predicted molecular weight of 39. 89 ku. We compared the E. ilishaeformis alignment of deduced amino acid sequences of G6Pase cDNA with Danio rerio, Mus musculu, Canis familiaris, Homo sapiens, Xenopns laevis, Sparus aurata and Tetraodon nigroviridis. The score was 95%, 63%, 63%, 63%, 63%, 69 %, 55% and 76% respectively. E. ilishaeformi G6Pase also contained three conserved domains. To examine the relationship between dietary carbohydrate and G6Pase gene expression, we compared the G6Pase mRNA levels in different dietary with or without carbohydrate. Two groups of Erythroculter ilishaeformis were pair-fed for 8 weeks either a high protein without carbohydrate （63.38% protein, 0 carbohydrate） or a low protein with carbohydrate （40. 53% protein, 23. 98% carbohydrate） diet. Real time RT-PCR was used to examine the levels of G6Pase mRNA in fasted and 3, 6, 12, 24 h after feeding in contrast to the different dietary. There was some increase in the level of G6Pase mRNA in dietary with and without carbohydrates at 12 h after feeding, which showed that refeeding affected the gene expression of G6Pase. Levels of G6Pase mRNA were 2 -4 times in dietary with carbohydrate than those without carbohydrate at fasted and 3, 6, 12 h after feeding. These results showed that dietary carbohydrate can enhance the liver G6Pase gene expression.