半滑舌鳎乙酰辅酶A羧化酶α基因全长cDNA分子克隆及饲料脂肪水平对其在肝脏中表达的影响

Full Length cDNA Molecular Cloning of Acetyl-CoA Carboxylase αGene and Effects of Dietary Lipid Level on Its Expression in the Liver of Half-Smooth Tongue Sole(Cynoglossus semilaevis)

本试验采用反转录PCR(RT-PCR)和c DNA末端快速扩增(RACE)技术从半滑舌鳎肝脏中克隆了乙酰辅酶A羧化酶α(ACC1)基因全长c DNA,并采用实时荧光定量PCR方法对半滑舌鳎ACC1基因在肠道、肝脏、肌肉、卵巢、脾脏、全脑、肾脏、心脏、肠系膜脂肪等组织中的表达进行了研究;此外,还研究了饲料脂肪水平对半滑舌鳎肝脏中ACC1基因表达的影响。结果表明:1)半滑舌鳎ACC1基因c DNA全长7 811 bp,含1个7 074 bp的开放阅读框,编码2 357个氨基酸,ACC1蛋白计算分子质量为266 ku,等电点为6.42。半滑舌鳎ACC1氨基酸序列保守位点包括1个ATP结合位点(Gly^(316)~Gly^(321))、1个生物素结合位点(Val^(785)-Met^(786)-Lys^(787)-Met^(788))、1个辅酶A结合位点(Ser^(1969)~Val^(1995))。此外,半滑舌鳎ACC1基因存在可变剪接,形成另外2个同工型(isoforms),与分子质量为266 ku的ACC1相比,分别少8和15个氨基酸。2)半滑舌鳎所有组织中均检测到ACC1基因的表达,肝脏和全脑中ACC1 mRNA相对表达量显著高于其他组织(P<0.05),分别为2.67和2.53;肠道、肠系膜脂肪和卵巢中次之,分别为1.14、1.10和0.97;肾脏中最低,仅为0.48。3)与对照组(未添加鱼油组)相比,3.5%鱼油组肝脏中A CC1 mRNA相对表达量显著降低(P<0.05);7.0%和10.0%鱼油组肝脏中A CC1 mRNA相对表达量进一步降低,显著低于3.5%组和对照组(P<0.05),同时10.0%鱼油组低于7.0%鱼油组(P>0.05)。综上,本试验克隆出了半滑舌鳎ACC1基因的全长c DNA,并得出半滑舌鳎ACC1蛋白的主要功能位点为ATP结合位点、生物素结合位点、辅酶A结合位点,与其他脊椎动物相比基本保守。半滑舌鳎ACC1基因主要在肝脏和全脑等生脂组织中表达,饲料中添加鱼油显著抑制其肝脏中ACC1基因的表达,且抑制作用与鱼油添加量呈正相关。

The full-length c DNA of acetyl-coa carboxylase α（ ACC1） gene was cloned from liver of half-smooth tongue sole（ Cynoglossus semilaevis） by reverse transcription PCR（ RT-PCR） and rapid amplification of c DNA ends（ RACE） methods. The expression of ACC1 mRNA in gut,liver,muscle,ovary,spleen,brain,kidney,heart,mesenteric adipose tissue of half-smooth tongue sole was analyzed by real-time fluorescence quantitative PCR（ RT-q PCR） method. In addition,the effects of dietary lipid level on ACC1 gene expression in liver of half-smooth tongue sole were investigated. The results show ed as follow s： 1） the full-length c DNA of ACC1 gene was 7 811 bp with a 7 074 bp open reading frame encoding 2 357 amino acids. The ACC1 protein has a calculated molecular weight of 266 ku and isolectric point of 6. 42. Some conserved sites of ACC1 amino acid sequence were found,including a ATP-binding site（ Gly^（316） to Gly^（321））,a biotin-binding site（ Val^（785）-Met^（786）-Lys^（787）-Met^（788））,and a Co A-binding site（ Ser^（1969） to Val^（1995））. In addition,the alternative splice varieties were found in ACC1 gene,and we present evidence for the presence of tw o isoforms of ACC1 in half-smooth tongue sole liver that differ from the 266 ku ACC1 by the absence of 8 and 15 amino acids. 2） The expression of ACC1 mRNA was detected in all examined tissues. The relative expression level of ACC1 mRNA in liver and brain were 2. 67 and 2. 53,respectively,which were significantly higher than that in other tissues（ P〈0. 05）; the relative expression level of ACC1 mRNA in gut,mesenteric adipose and ovary were second,which were 1. 14,1. 10 and0. 97,respectively; the relative expression level of ACC1 mRNA in kidney was the low est,only was 0. 48. 3）Compared with the control group（ without fish oil group）,diet added with 3. 5% fish oil significantly decreased the relative expression level of ACC1 mRNA in liver（ P〈0. 05）; diet added with 7. 0% and 10. 0% fish oil led to further decrease the relative expression level of ACC1 mRNA in liver,and it was significantly low er than that in 3. 5% fish oil group and control group（ P〈0. 05）,meanw hile,the 10. 0% fish oil group was low er than 7.0% fish oil group（ P〉0. 05）. In summary,we have cloned the full-length c DNA of ACC1 gene from half-smooth tongue sole. Compared with other vertebrates,the main functional sites（ ATP-binding site,biotin-binding site and Co A-binding site） are basically conserved. The lipogenic tissues,such as liver and brain,are the main ACC1 gene expressing tissues in half-smooth tongue sole. Moreover,liver ACC1 mRNA expression is inhibited after the fish fed diets added with fish oil,and the inhibitory effect is positively correlated with the addition of fish oil.