猪SCD1和SCD5双基因敲除细胞系的构建及其对脂肪沉积的影响
Construction of Porcine(Sus scrofa)SCD1 and SCD5 Double Knockout Cell Lines and Their Effects on Fat Deposition
方钱海 1郭帅 2任红艳 3白文哲 1高斯 3刘雯雯 3陈洪波 4张立苹3
作者信息
- 1. 湖北省农业科学院畜牧兽医研究所/动物胚胎工程与分子育种湖北省重点实验室,武汉 430064;武汉轻工大学动物科学与营养工程学院/湖北省家畜种业技术创新中心,武汉 430023
- 2. 武汉纽利特生物技术有限公司,武汉 430063
- 3. 湖北省农业科学院畜牧兽医研究所/动物胚胎工程与分子育种湖北省重点实验室,武汉 430064
- 4. 武汉轻工大学动物科学与营养工程学院/湖北省家畜种业技术创新中心,武汉 430023
- 折叠
摘要
脂肪沉积及脂肪酸组成对猪肉品质具有决定性影响,硬脂酰辅酶A去饱和酶(stearoyl-CoA desaturase,SCD)对脂肪酸组成及代谢具有重要的调控作用.目前SCD1和SCD5是包括人类(Homo sapiens)在内的多种脊椎动物中仅有的两种SCD亚型,为明确SCD1和SCD5对猪(Sus scrofa)脂肪酸种类与组成的影响及其作用机制,本研究利用单single guide RNA(sgRNA)和双sgRNA相结合介导的CRISPR/Cas9技术对SCD1及SCD5基因进行定点敲除,筛选获得纯合缺失单克隆细胞系,对其进行脂肪酸含量的检测分析,并检测双基因缺失对脂肪沉积和脂质代谢的影响.结果显示,SCD1及SCD5双基因编辑效率超过70%;SCD1及SCD5双基因缺失降低了其介导的限速反应产物棕榈油酸(C16∶1)和油酸(C18∶1)的相对含量;同时,细胞中多不饱和脂肪酸(polyunsaturated fatty acid,PUFA)的含量显著提高.SCD1和SCD5的缺失还影响了芥酸(C22∶1n9)、二十二碳五烯酸(docosapentaenoic acid,DPA)、二十二碳六烯酸(docosahexaenoic acid,DHA)、二十碳五稀酸(eicosapentaenoic acid,EPA)和亚油酸(C18∶2n6)等脂肪酸的含量;SCD1和SCD5整体缺失后甘油三酯的含量显著降低,影响了脂肪酸合成酶(fatty acid synthase,FASN)、酰基辅酶A合成酶3(acyl-CoA synthetase long-chain family member 3,ACSL3)等与脂肪酸和脂质代谢相关蛋白的表达.结果表明,SCD对脂肪酸的组成及代谢和脂肪沉积具有重要的调控作用.本研究为猪肉品质改良及肥胖疾病研究提供了细胞模型和参考.
Abstract
Fat deposition and fatty acid composition have a decisive impact on pork quality.Stearoyl CoA desaturase(SCD)plays an important regulatory role in fatty acid composition and metabolism.SCD1 and SCD5 are the only 2 subtypes of the SCD family in various vertebrates currently,including humans(Homo sapiens).In order to clarify the effects of SCD1 and SCD5 on the types and composition of fatty acids in pigs(Sus scrofa)and their mechanisms,this study utilized CRISPR/Cas9 technology mediated by a combination of single and double single guide RNA(sgRNA)to perform targeted knockout of SCD1 and SCD5 genes.Homozygous deletion monoclone cell lines were screened,and the fatty acid content was detected and analyzed,their effects on fat deposition and lipid metabolism were examined.The results showed that the efficiency of SCD1 and SCD5 double genome editing exceeded 70%;Moreover,the deletion of both SCD1 and SCD5 genes reduced the relative content of palmitoleic acid(C16∶1)and oleic acid(C18∶1)in the rate limiting reaction products mediated by them;Moreover,the content of polyunsaturated fatty acids(PUFAs)in cells increased significantly.The absence of SCD1 and SCD5 also affects the content of fatty acids such as erucic acid(C18∶2n6),docosapentaenoic acid(DPA),docosahexenoic acid(DHA),eicosapentaenoic acid(EPA)and linoleic acid;The overall absence of SCD1 and SCD5 also significantly reduced the content of triglycerides,affecting the expression of factors related to fatty acid and lipid metabolism,such as fatty acid synthase(FASN)and acyl-CoA synthatase long-chain family member 3(ACSL3).The results indicate that SCD might play an important regulatory role in the composition,metabolism,and deposition of fatty acids.This study provides a research model and reference for the improvement of pork quality and the study of obesity diseases.
关键词
CRISPR/Cas9技术/硬脂酰辅酶A去饱和酶(SCD)/脂肪酸代谢/脂肪沉积/多基因编辑Key words
CRISPR/Cas9 technology/Stearoyl-CoA desaturase(SCD)/Fatty acid metabolism/Fat deposition/Multi-gene editing引用本文复制引用
基金项目
国家重点研发计划(2021YFA0805901-4)
国家重点研发计划(NK2022010207)
湖北省农业科技创新中心项目(2023-620-000-001-20)
湖北省重点研发计划(2021BBA221)
动物胚胎工程及分子育种湖北省重点实验室开放课题(KLAEMB-2020-03)
武汉市生物育种关键技术攻关及新品种培育科技重大专项(2022021302024853)
出版年
2024
国家科技期刊平台
NSTL
万方数据