草鱼乙酰辅酶A羧化酶β基因全长cDNA分子克隆与表达分析

摘要

本试验采用反转录PCR(RT-PCR)和cDNA末端快速扩增(RACE)技术克隆获得草鱼乙酰辅酶A羧化酶β(ACC2)基因全长cDNA,并采用实时荧光定量PCR技术研究ACC2基因在肝胰脏、脾脏、大脑、前肠、中肠、后肠、肾脏、肌肉、心脏和肠系膜脂肪等组织中的表达,同时,还对饲喂不同脂肪源饲料12周后草鱼肝胰脏和肌肉中以及饥饿再次投喂后3、6、12和24 h肝胰脏中ACC2 mRNA的表达变化进行了研究.结果显示:草鱼ACC2基因cDNA全长7533 bp,含1个7149 bp的开放阅读框,编码2382个氨基酸,ACC2蛋白计算分子质量为268.34 ku,等电点为6.13.草鱼ACC2基因存在可变剪接,形成另外1个同工型(isoforms),比分子质量为268.34 ku的ACC2蛋白少8个氨基酸.ACC2基因在所有检测组织中均有表达,ACC2 mRNA的相对表达量在肌肉中最高,为29.13,在肝胰脏中最低,仅为1.90.肌肉中ACC2 mRNA的相对表达量与大脑、前肠和心脏差异不显著(P>0.05),但显著高于其他组织(P<0.05).投喂不同脂肪源饲料对草鱼肝胰脏及肌肉中ACC2 mRNA相对表达量无显著影响(P>0.05);饥饿再投喂后草鱼肝胰脏中ACC2 mRNA相对表达量在12 h达到高峰值,为6.17,之后明显下降,24 h时仅为2.84.本试验成功克隆了草鱼ACC2基因全长cDNA,其主要的功能位点ATP结合位点、生物素结合位点与其他脊椎动物相比基本保守.草鱼ACC2基因主要在肌肉等脂肪分解活跃的组织中表达,投喂不同脂肪源饲料对草鱼肝胰脏中ACC2 mRNA的相对表达量无显著影响;饥饿再投喂后,肝胰脏中ACC2 mRNA的相对表达量在投喂12 h后最高.%The full-length cDNA of acetyl-CoA carboxylase β(ACC2)gene was cloned from grass carp (Ctenopharyngodon idella)by reverse transcription PCR(RT-PCR)and rapid amplification of cDNA ends (RACE)methods. The expression of ACC2 gene in the hepatopancreas, spleen, brain, anterior intestine, mid-intestine, posterior intestine, kidney, muscle, heart and adipose tissues of grass carp was analyzed by quantitative real-time PCR method. Meanwhile,the ACC2 gene expression in the hepatopancreas and muscle of grass carp fed with diets containing different lipid sources for 12 weeks was studied. In addition, ACC2 gene expression in the hepatopancreas of grass carp at 3,6,12 and 24 h starvation and refeeding was also investiga-ted. The results showed that the full-length cDNA of ACC2 gene was 7 533 bp with a 7 149 bp open reading frame encoding 2 382 amino acids. The calculated molecular weight of ACC2 protein was 268.34 ku and the i-soelectric point was 6.13. In addition, alternative splicing was observed in ACC2 gene of grass carp and one isoform was formed, where eight amino acids were absent compared with that of the 268.34 ku ACC2 protein. The expression of ACC2 gene was detected in all examined tissues. The highest expression level of ACC2 mR-NA was found in the muscle(29.13), which was significantly higher than that in other tissues except for brain, anterior intestine and heart(P<0.05), whereas its expression in the hepatopancreas(only 1.90)was the lowest. There were no significant differences in ACC2 mRNA expression level in the hepatopancreas and muscle of grass carp after fed with diets containing different lipid sources(P>0.05). The highest expression level of ACC2 mRNA(6.17)in the hepatopancreas of grass carp was detected in the group of 12 h starvation and refeeding(P<0.05), which then decreased obviously and declined to 2.84 in the group of 24 h starvation and refeeding. In summary, we have cloned the full-length cDNA of ACC2 gene from grass carp. Compared with other vertebrates, the main functional sites(ATP-binding site and biotin-binding site)are basically con-served. The active lipolysis tissues, such as muscle, are the main ACC2 gene expressing tissues in grass carp. Moreover, the hepatopancreas ACC2 mRNA expression level is not affected after the grass carp fed with diets containing different lipid sources, and the highest expression level of ACC2 mRNA in the hepatopancreas of grass carp is detected when 12 h starvation and refeeding.