FADS1基因对猪不饱和脂肪酸含量的影响研究

doi:10.16431/j.cnki.1671-7236.2023.02.003

中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (2): 451-459.doi: 10.16431/j.cnki.1671-7236.2023.02.003

FADS1基因对猪不饱和脂肪酸含量的影响研究

张天天^1,2, 华再东², 任红艳², 顾浩², 周彬¹, 毕延震²

1. 湖北工业大学生物工程与食品学院, 发酵工程教育部重点实验室, 武汉 430068;
2. 湖北省农业科学院畜牧兽医研究所, 动物胚胎工程及分子育种湖北省重点实验室, 武汉 430064

收稿日期:2022-07-19 出版日期:2023-02-05 发布日期:2023-02-06
通讯作者: 周彬, 毕延震 E-mail:zhoubin4111@163.com;sukerbyz@126.com
作者简介:张天天,E-mail:1760200256@qq.com
基金资助:
湖北省重点研发计划(青年科学家项目)(2021BBA221);湖北省农业科技创新中心项目(2022-620-000-001-20);湖北省农业科学院领军人才(L2018015)

Effect of FADS1 Gene on Unsaturated Fatty Acid Synthesis

ZHANG Tiantian^1,2, HUA Zaidong², REN Hongyan², GU Hao², ZHOU Bin¹, BI Yanzhen²

1. Key Laboratory of Fermentation Engineering, Ministry of Education, College of Biological Engineering and Food, Hubei University of Technology, Wuhan 430068, China;
2. Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Sciences, Wuhan 430064, China

Received:2022-07-19 Online:2023-02-05 Published:2023-02-06

摘要/Abstract

摘要： 【目的】研究脂肪酸去饱和酶1(fatty acid desaturases 1,FADS1)基因对不饱和脂肪酸代谢的调控作用,为揭示其分子机制提供参考。【方法】利用PCR扩增猪FADS1基因的CDS区,将目的基因与pcDNA3.1(-)骨架载体连接获得重组表达载体pcDNA3.1-FLAG-FADS1,将重组表达载体瞬时转染宁乡猪肾成纤维细胞,转染后24、48 h分别收集细胞,利用实时荧光定量PCR和Western blotting检测FADS1基因的表达情况,经过遗传霉素(geneticin,G418)筛选之后获得稳定表达FADS1基因的细胞系,命名为1-4^#。利用甘油三酯检测试剂盒检测野生型和1-4^#细胞甘油三酯含量变化;利用气相色谱质谱法(gas chromatography-mass spectrometer,GC-MS)检测野生型和1-4^#细胞不饱和脂肪酸含量变化情况。【结果】成功获得过表达载体pcDNA3.1-FLAG-FADS1,在转录水平和蛋白水平检测均有过表达效果。通过G418筛选出1株稳定过表达FADS1基因的单克隆细胞。甘油三酯含量测定结果显示,与野生型宁乡猪肾成纤维细胞相比,过表达FADS1的宁乡猪肾成纤维细胞中甘油三酯的含量显著降低(P＜0.05)。不饱和脂肪酸检测结果显示,过表达FADS1后,总脂肪酸的含量极显著升高(P＜0.01),提高1.8倍,多不饱和脂肪酸(polyunsaturated fatty acid,PUFA)中ω-3 PUFA、ω-6 PUFA含量均极显著升高(P＜0.01),二十碳五烯酸(eicosapentaenoic acid,EPA)、二十二碳五烯酸(docosapentaenoic acid,DPA)、二十二碳六烯酸(docosahexaenoic acid,DHA)的相对含量极显著提高(P＜0.01),ω-3/ω-6值显著提高(P＜0.05)。【结论】成功构建超表达载体pcDNA3.1-FLAG-FADS1,并在宁乡猪肾成纤维细胞中成功筛选到了1株稳定超表达FADS1基因的单克隆细胞1-4^#,经检测,FADS1基因超表达能显著降低细胞中甘油三酯含量、提高ω-3 PUFA含量及占比,提高ω-3/ω-6值。

关键词: FASD1基因; 过表达; 脂肪酸; ω-3; ω-6

Abstract: 【Objective】 To study the regulatory effect of fatty acid desaturases 1 (FADS1) gene on the metabolism of unsaturated fatty acids, and provide a reference for revealing its molecular mechanism. 【Method】 The FADS1 gene was overexpressed in the primary fetal fibroblasts isolated by vector pcDNA3.1-FLAG-FADS1, which was designed according to the CDS sequence of FADS1 gene. The constructed vector was transferred into the cells and screened by geneticin (G418) to obtain monoclonal cell lines. After transfection, cells were collected at 24 and 48 h respectively, and the expression of FADS1 gene was detected by Real-time PCR and western blotting, and the cell line with stable expression of FADS1 gene was obtained after G418 screening, named 1-4^#. These cells were used to detect the change of triglyceride content and analysis of unsaturated fatty acid composition through GC-MS.【Result】 The results showed that the overexpression vector pcDNA3.1-FLAG-FADS1 was successfully obtained, and the overexpression effect was detected at both the transcription level and the protein level. Then these cells were cultured in complete medium with G418 to obtain stable cell lines. Triglyceride assay showed that FADS1 gene overexpression could reduce the content of triglyceride in primary fetal fibroblasts (P＜0.05). The test results of unsaturated fatty acids showed that after overexpression of FADS1, the content of total fatty acids increased extremely significantly (P＜0.01) and increased by 1.8 times. In polyunsaturated fatty acids (PUFA), the contents of ω-3 PUFA and ω-6 PUFA were extremely significantly increased (P＜0.01), the relative contents of eicosapentaenoic acid(EPA), docosapentaenoic acid(DPA) and docosahexaenoic acid(DHA) were extremely increased (P＜0.01), and the ratio of ω-3/ω-6 was significantly increased (P＜0.05).【Conclusion】 In summary, the overexpression vector pcDNA 3.1-FLAG-FADS1 was successfully constructed, and a single clonal cell with stable hyperexpression of FADS1 gene was successfully screened in primary fetal fibroblasts, FADS1 gene could significantly increase the content of ω-3 PUFA and increase the ratio of ω-3/ω-6.

Key words: FADS1 gene; overexpression; fatty acids; ω-3; ω-6

中图分类号:

S828

张天天, 华再东, 任红艳, 顾浩, 周彬, 毕延震. FADS1基因对猪不饱和脂肪酸含量的影响研究[J]. 中国畜牧兽医, 2023, 50(2): 451-459.

ZHANG Tiantian, HUA Zaidong, REN Hongyan, GU Hao, ZHOU Bin, BI Yanzhen. Effect of FADS1 Gene on Unsaturated Fatty Acid Synthesis[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 451-459.

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FADS1基因对猪不饱和脂肪酸含量的影响研究

Effect of FADS1 Gene on Unsaturated Fatty Acid Synthesis

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