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

发布时间：2018-12-28 19:07

【摘要】：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(P0.05),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(P0.05);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(P0.05),同时10.0%鱼油组低于7.0%鱼油组(P0.05)。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。
[Abstract]:In this study, the full length c DNA, of acetyl coA carboxylase 伪 (ACC1) gene was cloned from liver of Cynoglossus semilaevis by reverse transcription PCR (RT-PCR) and rapid amplification of (RACE) at the end of c DNA. The expression of ACC1 gene in intestine, liver, muscle, ovary, spleen, whole brain, kidney, heart and mesenteric fat was studied by real-time fluorescence quantitative PCR. In addition, the effect of dietary fat level on the expression of ACC1 gene in liver of Cynoglossus semilaevis was studied. The results showed that: 1) c DNA of Cynoglossus semilaevis ACC1 gene had a 7 811 bp, open reading frame of 7 074 bp, encoding 2 357 amino acids, and the calculated molecular weight of ACC1 protein was 266 ku, isoelectric point 6.42. The conserved amino acid sequence of ACC1 of Cynoglossus semilaevis includes one ATP binding site (Gly~ (316) ~ Gly~) (321),) and one biotin binding site (Val~ (785) -Met~ (786) -Lys~ (787) -Met~ (788),). A CoA binding site (Ser~ _ (1969) ~ Val~ (1995).) In addition, there was variable splicing in ACC1 gene of Cynoglossus semilaevis. The expression of ACC1 gene was detected in all tissues of Cynoglossus semilaevis compared with ACC1 with molecular weight of 266 ku. The relative expression of ACC1 mRNA in liver and brain was significantly higher than that in other tissues (P0.05), which were 2.67 and 2.53 respectively. The intestinal tract, mesenteric fat and ovary were 1.141.10 and 0.97 respectively. Compared with the control group (no fish oil group), the relative expression of A CC1 mRNA in the liver of 3.5% fish oil group was significantly lower than that in the control group (P 0.05). The relative expression of A CC1 mRNA in the liver of 7.0% and 10.0% fish oil groups was significantly lower than that of 3.5% group and control group (P0.05), while that of 10.0% fish oil group was lower than that of 7.0% fish oil group (P0.05). In conclusion, the full-length c DNA, of ACC1 gene of Cynoglossus semilaevis was cloned and the main functional sites of ACC1 protein were ATP binding site, biotin binding site and coenzyme A binding site, which were conserved compared with other vertebrates. The ACC1 gene of Cynoglossus semilaevis was mainly expressed in lipids and lipids of the whole brain. Fish oil supplementation significantly inhibited the expression of ACC1 gene in the liver, and the inhibitory effect was positively correlated with the amount of fish oil added.
【作者单位】：淮海工学院;江苏省海洋生物产业技术协同创新中心;浙江省淡水水产研究所;
【基金】：国家自然科学基金(31272636) 江苏省高校自然科学研究重大项目(10KJA240002) 江苏省自然科学基金(BK2012664) 江苏省海洋生物技术重点实验室开放基金(2009HS15) 浙江省重大科技专项(2012C12907-2) 江苏省优势学科建设工程项目(PAPD) 国家科技支撑计划(2012BAD26B04-04)
【分类号】：S917.4

【相似文献】