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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。
In this study, the full-length c DNA of ACC1 gene was cloned from the livers of C. halophila using reverse transcription-polymerase chain reaction (RT-PCR) and rapid c DNA end-amplification (RACE) PCR method was used to study the expression of ACC1 gene in the tissues of intestinal tract, liver, muscle, ovary, spleen, whole brain, kidney, heart and mesenteric fat. The impact of expression. The results showed as follows: 1) The c DNA of ACC1 gene was 7 811 bp in length and contained 1 open reading frame of 7 074 bp, encoding 2 357 amino acids. The ACC1 protein had a molecular mass of 266 ku and an isoelectric point of 6.42. The conserved amino acid sequences of ACC1 in the half-smooth tongue sole include one ATP binding site (Gly ~ 316 ~ Gly ~ 321) and one biotin binding site (Val ~ (785) -Met ~ (786) -Lys ~ (787) -Met ~ (788)) and one coenzyme A binding site (Ser ~ (1969) ~ Val ~ (1995)). In addition, there is an alternative splicing of the ACC1 gene in C. sinensis, forming two more isoforms, which are 8 and 15 amino acids less, respectively, compared to the 266 amino acid molecule of ACC1. 2) The expression of ACC1 gene was detected in all tissues of the half-smooth tongue ACC. The relative expression of ACC1 mRNA in the liver and whole brain was significantly higher than that in other tissues (P <0.05), which were 2.67 and 2.53, respectively. The intestinal, mesenteric fat and ovary (1.14, 1.10 and 0.97, respectively), and the lowest in kidney was only 0.48. 3 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 (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 in 3.5% and control groups (P <0.05) ). In conclusion, we cloned the full-length cDNA of ACC1 gene of C. sinensis and found that the main functional sites of ACC1 protein in A. paratyphus were ATP-binding site, biotin-binding site, coenzyme A binding site and other vertebrates Compared to the basic conservative. The ACC1 gene was mainly expressed in adipose tissue such as the liver and whole brain. The addition of fish oil significantly inhibited the ACC1 gene expression in the liver, and the inhibitory effect was positively correlated with the addition of fish oil.