奶山羊超长链脂肪酸延伸酶6基因(ELOVL6)的克隆及其对4个脂肪酸代谢基因表达水平的影响  

Cloning of Elongase of Very Long Chain Fatty Acids 6 Gene(ELOVL6)and Its Effect on Four Genes Expression Involved in Fatty Acid Metabolism in Dairy Goat(Capra hircus)

作　　者：吴敏[1] 罗军[1] 朱江江[1] 姚大为[1] 李君[1] 孙雨婷[1] 史怀平[1] 

机构地区：[1]西北农林科技大学动物科技学院/陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2014年第6期672-680,共9页Journal of Agricultural Biotechnology

基　　金：公益性行业(农业)科研专项(No.201103038);国家自然科学基金项目(No.31372281);国家转基因新品种培育重大专项(2014ZX08009-051B)

摘　　要：超长链脂肪酸延伸酶6(elongase of very long chain fatty acids 6,ELOVL6)是长链脂肪酸延长反应的限速酶。本研究旨在通过对奶山羊(Capra hircus)ELOVL6基因的克隆、组织表达分析以及腺病毒(Adenovirus)介导的超表达技术,研究该基因超表达后对乳腺上皮细胞中脂肪酸代谢相关基因的影响。利用RT-PCR技术克隆了奶山羊ELOVL6基因的CDS区;利用实时荧光定量PCR(qRT-PCR)检测该基因在泌乳期奶山羊10个组织中的相对表达量;构建该基因的重组腺病毒超表达载体,包装出高滴度的腺病毒并感染奶山羊原代乳腺上皮细胞,qRT-PCR检测ELOVL6超表达效果及其对脂肪酸代谢相关基因的影响。结果表明,ELOVL6基因CDS区序列长度为795 bp(GenBank登录号:KF667508),共编码264个氨基酸。同源分析发现,奶山羊ELOVL6基因CDS区核酸序列与牛(Bos taurus)、大鼠(Rattus norvegicus)和人(Homo sapiens)的同源性分别为97%、90%和93%,氨基酸序列同源性分别为99%、92%、95%。利用TMpred对ELOVL6蛋白跨膜结构预测分析,发现该蛋白有5个跨膜区,保守的组氨酸模体(HXXHH)位于ELOVL6蛋白的第二与第三跨膜螺旋间。ELOVL6蛋白质疏水性预测显示,该蛋白总体具有较强的疏水性。组织表达分析显示,ELOVL6基因在脂肪组织中表达量最高(P<0.01),小肠次之(P<0.01),心脏中表达量最低。超表达ELOVL6基因的重组腺病毒的滴度为108U/mL,病毒液感染原代乳腺上皮细胞48 h后,ELOVL6基因的表达量上升约200倍;脂肪酸代谢相关基因检测分析发现,固醇调节元件结合蛋白-1基因(SREBP-1)的表达量显著下降(P<0.05),脂肪分化相关蛋白基因(ADRP)的表达量显著升高(P<0.05),过氧化物酶体增殖物激活受体γ基因(PPARγ)及脂肪酸转位酶基因(CD36)的表达量无明显变化。研究结果表明,ELOVL6基因可以影响奶山羊乳腺上皮细胞脂肪酸代谢相关基因的表达,对乳腺脂肪酸代谢具有一定的调控作用。本研究为EElongase of very long chain fatty acids 6 （ELOVL6） is the rate-limiting enzyme in the elongationcycle in mammals. In this study, ELOVL6 coding sequences （CDS） was cloned by RT-PCR, tissue expession was analyzed by Real-time quantitative PCR （qRT-PCR） and constructed a recombinant Adenovirusoverexpression vector for ELOVL6 of dairy goat （Capra hircus） to investigate its effect of related genes on fatty acid metabolism after infecting recombinant Adenovirus in dairy goat mammary epithelial cells（GMEC）. The results showed that the CDS of ELOVL6 gene was 795 bp （GenBank No. KF667508）, encoding 264 amino acids. Compared with cattle（Bos taurus）, rat（Rattus norvegicus） and human （Homo sapiens）, thenucleic acid sequence homology of dairy goat ELOVL6 was 97%, 90% and 93%, and the amino acid sequence homology was 99%, 92% and 95%, respectively. The protein structure analysis using TMpred showed that 5transmembrane helixes were speculated in ELOVL6 protein, and there was a conserved histidine box （HWYHH） between the second and the third transmembrane helix. The ELOVL6 protein had stronghydrophobic properties analysed by ProtScale. Tissue expression analysis showed that ELOVL6 gene was themost abundant in adipose tissue（P〈0.01）, followed by small intestine（P〈0.01）, and minimum in heart. Thetiter of recombinant Adenovirus containing ELOVL6 gene was 10^8 U/mL. Compared with Ad- GFP group, ELOVL6 mRNA level increased by about 200-fold in in GMEC after incubated with the virus for 48 h. Sterolregulatory element binding protein-1 （SREBP-1） was decreased significantly （P〈0.05）, and adipose differentiation related protein （ADRP） was increased significantly （P〈0.05）. There was no significant effect onthe expression of peroxisome proliferator activated receptor γ （PPARγ） and fatty acid translocase （CD36）. These results indicated that ELOVL6 can influence the expression of genes related to fatty acid metabolism,and may have certain function on fatty acid

关 键 词：奶山羊 超长链脂肪酸延伸酶6(ELOVL6) 组织表达 超表达 

分 类 号：S565.01[农业科学—作物学]