小尾寒羊ACAA1基因CDS克隆及组织表达谱分析

Cloning and Tissue Expression Profile Analysis of ACAA1 Gene CDS in Small-tail Han Sheep

为探索乙酰辅酶A酰基转移酶1(acetyl-coenzyme A acyltransferase 1,ACAA1)基因的生物学功能,明确该基因在小尾寒羊不同组织中的表达差异,试验采集小尾寒羊心脏、肝脏、胃、十二指肠、小肠、背最长肌、皮下脂肪组织,提取总RNA,根据GenBank中公布的绵羊ACAA 1基因序列(登录号:XM_004018227.3)设计引物,利用RT-PCR和分子克隆技术获得小尾寒羊ACAA 1基因完整CDS,并对其进行生物信息学分析;利用实时荧光定量PCR检测该基因在小尾寒羊不同组织中的表达差异。结果显示,小尾寒羊ACAA 1基因CDS长为1 071 bp,编码356个氨基酸;小尾寒羊ACAA 1基因氨基酸序列与绵羊、山羊同源性最高,约为100%,与食蟹猴、狒狒和野猪的同源性均为93.0%;系统进化树结果显示,小尾寒羊与绵羊、山羊位于同一分支,亲缘关系较近,与穿山甲、狒狒的亲缘关系较远;各物种间同源性较高,保守性较强。ACAA1蛋白分子质量约为36.92 ku,分子式为C 1603 H 2638 N 458 O 501 S 18,理论等电点为7.48,半衰期为30 h,消光系数为9 440,稳定系数为40.88,脂溶系数为92.67,亲水性氨基酸残基约占58%,为不稳定的碱性脂溶性亲水蛋白;不存在跨膜结构与信号肽,不是分泌蛋白;主要分布在过氧化物酶体,存在25个磷酸化位点、3个潜在的N-糖基化位点和4个O-糖基化位点。ACAA1蛋白二级结构含有α-螺旋(37.92%)、β-折叠(12.64%)和无规则卷曲(49.44%),三级结构预测结果与其一致。实时荧光定量PCR结果显示,ACAA 1基因在小尾寒羊不同组织中均有表达,其中在肝脏、皮下脂肪、小肠中表达量相对较高。本试验结果为进一步探索小尾寒羊ACAA 1基因生物学功能及筛选与肉质性状相关的候选基因提供依据。

To explore the biological function of acetyl-coenzyme A acyltransferase 1(ACAA1)gene,and determine the expression difference of ACAA 1 gene in different tissues of Small-tail Han sheep,in this experiment,the total RNA was extracted from heart,liver,stomach,duodenum,small intestine,longissimus dorsi muscle and subcutaneous fat tissue of Small-tail Han sheep.Primers were designed based on the sequence of ACAA 1 gene in sheep published in GenBank(accession No.:XM_004018227.3).The complete CDS of ACAA 1 gene in Small-tail Han sheep was obtained by RT-PCR and molecular cloning technology,and bioinformatics analysis was carried out.Real-time PCR was used to detect the expression difference of ACAA 1 gene in different tissues of Small-tail Han sheep.The results showed that the length of ACAA 1 gene CDS in Small-tail Han sheep was 1 071 bp,encoding 356 amino acids,the amino acid sequence of ACAA 1 gene in Small-tail Han sheep had the highest homology with Ovis aries and Capra hircus,which were about 100%,and the homology with Macaca fascicularis,Theropithecus gelada and Sus scrofa were 93.0%.The results of phylogenetic tree showed that Small-tail Han sheep was located in a branch with sheep and goat,the relationship was closer,and the relationship with the Manis javanica and Theropithecus gelada was far away,the homology among the species was higher,and the conservation was stronger.The molecular weight of ACAA1 protein was 36.92 ku,the molecular formula was C 1603 H 2638 N 458 O 501 S 18,the theoretical isoelectric point was 7.48,the half-life was 30 h,the extinction coefficient was 9 440,the stability coefficient was 40.88,and the fat solubility coefficient was 92.67,the hydrophilic amino acid residue was about 58%,this protein was an unstable alkaline fat-soluble hydrophilic protein.There was no transmembrane structure and signal peptide,not secreted protein.It mainly distributed in peroxisomes,there were 25 phosphorylation sites,3 potential N-glycosylation sites and 4 O-glycosylation sites;The secondary structure of ACAA1 protein contained alpha helix(37.92%),beta fold(12.64%)and random coil(49.44%),which was consistent with the tertiary structural results.Real-time PCR results showed that ACAA 1 gene was expressed in different tissues of Small-tail Han sheep,and the expression levels were relatively higher in liver,subcutaneous fat and small intestine.This results provided a basis for further exploration of the biological function of ACAA 1 gene in Small-tail Han sheep and screening for candidate genes related to meat quality.