本试验采用简并引物反转录聚合酶链式反应(RT-PCR)和cDNA末端快速扩增(RACE)技术克隆西伯利亚鲟(Acipenser baerii)肝脏糖异生途径关键酶——C型和M型磷酸烯醇式丙酮酸羧基酶(PEPCK-C和PEPCK-M)、果糖-1,6-二磷酸酶(FBPase)和葡萄糖-6-磷酸酶(G6Pase)基因cDNA全长序列。结果显示:西伯利亚鲟PEPCK-C基因(GenBank登录号JQ995143)cDNA全长2 598 bp,开放阅读框1 869 bp,编码622个氨基酸,预测其分子质量为69.62 ku,与其他物种的相似性为77.3%~80.5%;PEPCK-M基因(GenBank登录号JQ995142)cDNA全长3 277 bp,开放阅读框1 935 bp,编码644个氨基酸,预测其分子质量为71.11 ku,与其他物种的相似性为64.6%~78.3%;FBPase基因(GenBank登录号JF834908)cDNA全长1 372 bp,开放阅读框1 017 bp,编码338个氨基酸,预测其分子质量为36.60 ku,与其他物种的相似性为73.4%~88.7%;G6Pase基因(GenBank登录号JF834907)cDNA全长2 625 bp,开放阅读框1 080 bp,编码359个氨基酸,预测其分子质量为40.62 ku,与其他物种的相似性为67.2%~72.7%。西伯利亚鲟糖异生途径关键酶基因全长cDNA序列的获得将为进一步研究其糖异生调控机理,比较其与典型肉食性鱼类和哺乳动物的异同奠定基础。
The full-length cDNAs of the key enzymes of gluconeogenesis pathway, including phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M), fructose-1,6-bisphosphatase (FBPase) and glucose-6-phosphatase (G6Pase) were cloned from Siberian sturgeon (Acipenser baerii) liver using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The results showed as follows: the full-length PEPCK-C cDNA of Siberian sturgeon (GenBank accession No. JQ995143) was 2 598 bp, consisting of an open reading frame (ORF) of 1 869 bp encoding a polypeptide of 622 amino acids with a theoretical molecular weight of 69.62 ku, and shared 77.3% to 80.5% similarity with other PEPCK-C genes. The full-length PEPCK-M cDNA of Siberian sturgeon (GenBank accession No. JQ995142) was 3 277 bp, consisting of an ORF of 1 935 bp encoding a polypeptide of 644 amino acids with a theoretical molecular weight of 71.11 ku, and shared 64.6% to 78.3% similarity with other PEPCK-M genes. The full-length FBPase cDNA of Siberian sturgeon (GenBank accession No. JF834908) was 1 372 bp, consisting of an ORF of 1 017 bp encoding a polypeptide of 338 amino acids with a theoretical molecular weight of 36.60 ku, and shared 73.4% to 88.7% similarity with other FBPase genes. The full-length G6Pase cDNA of Siberian sturgeon (GenBank accession No. JF834907) was 2 625 bp, consisting of an ORF of 1 080 bp encoding a polypeptide of 359 amino acids with a theoretical molecular weight of 40.62 ku, and shared 67.2% to 72.7% similarity with other G6Pase genes. The results of this study will provide a scientific basis for further study to explain the difference in the ability of dietary carbohydrate utilization and regulatory mechanism of gluconeogenesis in Siberian sturgeon compared with mammalian and carnivorous teleost.
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