草鱼脂酰辅酶A合成酶4基因全长cDNA分子克隆及表达调控

Full-Length cDNA Molecular Cloning and Expression Regulation of Acyl Coenzyme A Synthetase 4 Gene in Grass Carp

本试验采用反转录PCR(RT-PCR)技术克隆草鱼脂酰辅酶A合成酶4(ACSL4)基因全长cDNA,利用实时荧光定量PCR技术研究ACSL4基因在草鱼大脑、心脏、脾脏、肝胰脏、肌肉、肾脏、肠系膜脂肪、前肠、中肠、后肠10种组织中的表达情况,并对投喂不同淀粉源饲料以及饥饿再投喂0、3、6、12和24 h后草鱼肝胰脏中ACSL4基因表达情况进行研究。结果显示:草鱼ACSL4基因cDNA全长2 418 bp,其开放阅读框2 121 bp,共编码706个氨基酸;草鱼ACSL4蛋白的氨基酸序列包含1个跨膜结构域、1个脂肪酸绑定基序和1个ATP/AMP绑定基序。草鱼ACSL4 mRNA在大脑和脾脏中相对表达量较高,显著高于其他组织(P <0.05),在肌肉中相对表达量最低;投喂不同淀粉源饲料对草鱼肝胰脏中ACSL4 mRNA相对表达量无显著影响(P>0.05);饥饿再投喂12 h后,草鱼肝胰脏中ACSL4 mRNA相对表达量达到最高值,显著高于其他时间段(P<0.05),24 h后恢复至最初水平。本试验从草鱼10种混合组织中克隆了ACSL4基因全长cDNA,其所编码的氨基酸序列的主要功能性位点脂肪酸绑定基序和ATP/AMP绑定基序在不同物种间高度保守;草鱼ACSL4基因主要在大脑和脾脏中表达;饲料中淀粉源对草鱼肝胰脏中ACSL4基因的表达无显著影响;在饥饿再投喂12 h后,草鱼肝胰脏中ACSL4 mRNA相对表达量达到最高值,随后显著下降至最初水平。

The full-length cDNA of long chain acyl coenzyme A synthetase 4(ACSL4)gene was cloned from grass carp by reverse-transcription PCR(RT-PCR)technique.Tissue distribution of ACSL4 gene in the brain,heart,spleen,hepatopancreas,muscle,kidney,adipose tissues,anterior intestine,mid-intestine and posterior intestine of grass carp was analyzed by real-time quantitative PCR technique.Meanwhile,the ACSL4 gene expression in the hepatopancreas of grass carp fed with diets containing different starch sources was studied.In addition,the ACSL4 gene expression in the hepatopancreas of grass carp at 0,3,6,12 and 24 h after starvation and refeeding was also investigated.The results showed as follows:the full-length cDNA of ACSL4 gene was 2 418 bp with a 2 121 bp open reading frame(ORF)encoding 706 amino acids;the ACSL4 protein contained a trans-membrane domain,a ATP/AMP signature motif and a fatty acyl coenzyme A synthetase(FACS)signature motif.The relative expression level of ACSL4 mRNA in brain and spleen were higher,which were significantly higher than those in the other tissues(P<0.05),and the lowest in muscle.There were no significant differences of ACSL4 mRNA relative expression level in the hepatopancreas of grass carp after fed with diets containing different starch sources(P>0.05).However,the highest expression level of ACSL4 mRNA in the hepatopancreas of grass carp was detected after 12 h of starvation and refeeding,which was significantly higher than that at the other time(P<0.05),and then it returned to its beginning level after 24 h of starvation and refeeding.In summary,we have cloned the full-length cDNA of ACSL4 gene from grass carp,and the main functional sites ATP/AMP signature motif and FACS signature motif of amino acid sequences are both highly conserved in different species.Brain and spleen are the main ACSL4 gene expressing tissues in grass carp.Moreover,the ACSL4 gene expression in hepatopancreas of grass carp is not significantly affected by different dietary starch sources,and the highest relative expression level of ACSL4 mRNA in the hepatopancreas of grass carp is detected after 12 h of starvation and refeeding,and then it decreases significantly to its beginning level.