奶山羊SCD基因CDS区的克隆、序列分析及过表达

Cloning, Sequence Analysis and Over-Expression of SCD Gene of Dairy Goat

【目的】研究奶山羊硬脂酰辅酶A去饱和酶基因(stearoyl-CoA desaturase,SCD)在乳腺上皮细胞中对不饱和脂肪酸合成过程相关基因的调控及对细胞脂肪酸组成的影响。【方法】用RT-PCR方法从西农萨能羊乳腺组织中扩增SCD基因,进行序列分析和组织表达谱分析;构建重组腺病毒质粒,进行包装和扩增后,感染体外培养的奶山羊原代乳腺上皮细胞,实时定量检测相关基因的表达,western blotting检测蛋白变化,气相色谱-质谱联用分析细胞内脂肪酸组成变化。【结果】①获得了全长1 080 bp的山羊乳腺SCD基因CDS(GenBank登录号:GU947654)并对其进行了生物信息学分析;②奶山羊SCD基因在乳腺、肺和皮下脂肪组织中高表达,而在心脏、瘤胃和卵巢组织中的表达量较低;③成功构建了奶山羊SCD基因的重组腺病毒载体并制备出高滴度的重组病毒液,重组腺病毒感染原代乳腺上皮细胞后,检测结果表明细胞中SCD基因过表达极显著,FASN、H-FABP、LEPR和LXRα等基因表达下调,而PPARγ和LPL基因的表达量显著性增加;④棕榈酸和硬脂酸的含量下降,不饱和脂肪酸含量上升。【结论】SCD基因在奶山羊乳腺中发挥着重要作用,FASN、H-FABP、LEPR、LXRα、PPARγ和LPL基因的表达与其相关,这些基因共同影响乳脂中脂肪酸的组成。

【Objective】To study the function of stearoyl-CoA desaturase （SCD） gene in mammary gland fatty acid metabolism emphasizing on how it regulates genes involved in unsaturated fatty acid synthesis as well as its impact on fatty acid composition.【Method】The sequence of SCD in dairy goat was cloned using RT-PCR for bioinformatics and tissue mRNA expression analysis. A recombinant adenovirus vector was constructed to over-express the gene in cultured goat mammary epithelial cells. Changes of gene expression, protein yield and fatty acid contents after over-expression were detected with real-time fluorescence quantitative PCR, western blotting and GC-MS. 【Result】The CDS of goat SCD gene is 1080 bp in length, coding 359 amino acid residues （GenBank accession： GU947654）. The SCD gene was expressed higher in mammary gland, lung and subcutaneous fat, lower in heart, rumen and ovary. Recombinant adenovirus vector containing goat SCD open reading frame was constructed, packaged and amplified in HEK293. The gene expressions of FASN, A-FABP, LEPR and LXRα were down regulated after over-expression of SCD, while expressions of PPARγ and LPL were up regulated significantly. Synthesis of palmitic acid and stearic acid in the cells decreased, while unsaturated fatty acids increased.【Conclusion】 SCD gene plays an important role in goat mammary fatty acid metabolism, and a major role in unsaturated fatty acid synthesis by interaction with other genes including FASN, H-FABP, LEPR, LXRα, PPARγ and LPL.