摘要
【目的】ω-3多不饱和脂肪酸(PUFAs)对人类具有重要的营养价值与医疗作用。随着生物技术的发展,通过转基因技术可实现哺乳动物体内自身合成多不饱和脂肪酸,进而改变猪肉中不饱和脂肪酸的含量和种类,对人类的营养健康具有积极意义。【方法】克隆猪体内缺乏的ω-3多不饱和脂肪酸脱氢酶基因FAT1和Δ-12脂肪酸去饱和酶的关键基因FAT2,构建携带FAT1-FAT2双基因的多位点打靶载体。该载体以猪rRNA基因间的内部转录间隔序列为靶位点;为提高定点整合效率,引入NEO、TK正负筛选系统进行双重选择;加入EGFP报告基因和Cre/Loxp系统,便于筛选阳性同源重组细胞克隆及后期删除筛选基因。将所构建的携带FAT1-FAT2双基因的打靶载体,通过脂质体介导瞬时转染猪肾细胞PK15。【结果】RT-PCR结果显示,FAT1-FAT2双基因在猪肾细胞PK15中实现表达;脂肪酸测定结果显示,ω-6 PUFAs、ω-3PUFAs在对照组PN1(转染对照质粒PN1)、转染试验组PF1(转染携带FAT1基因)、转染试验组PF1F2(转染携带FAT1-FAT2双基因)中的含量分别为7.9%、7.03%、3.92%和5.64%、7.01%、9.43%,差异均显著;ω-6/ω-3比例由对照组的1.4显著下降到试验组的0.42~1。【结论】构建携带FAT1-FAT2双基因的打靶载体转染猪肾细胞PK15可以表达产生FAT1-FAT2 mRNA,且可以显著提高转染细胞中ω-3 PUFAs的含量,为下一步生产转FAT1-FAT2双基因猪奠定基础。
【Objective】Polyunsaturated fatty acids(PUFAs)play a vital role in human medicine and nutritional value.With the development of biotechnology,it is possible to change the variety and content of unsaturated fatty acid in pork by transgenic technology to synthesize PUFAs by mammalian itself,which contribute to the human nutrition and health.【Method】By cloning FAT1 and FAT2 genes that modulatedω-3 polyunsaturated fatty acid dehydrogenase andΔ-12 fatty acid desaturase which were lack in pigs,a mutiple loci targeting vector that carrying the FAT1-FAT2 gene was constructed.This vector used the internal transcribe spacer sequences(ITS)that between the rRNA genes of pig as the target loci.To improve the efficiency of targeted integration,the positive and negative screening system of NEO and TK was introduced into the vector for double selection.EGFP reporter gene and Cre/Loxp system were added to the vector to screen out the positive clones of homologous recombination cells.The targeting vector with the FAT1-FAT2 gene was transfected into porcine kidney cell PK15 by liposome.【Result】RT-PCR results showed that FAT1-FAT2 gene expressed in porcine kidney cell PK15.The GC-MS results showed that the contents ofω-6 PUFAs andω-3PUFAs in control group PN1(cells that transfected with PN1 plasmid),treatment group PF1(cells that transfected with FAT1 gene),treatment group PF1F2(cells that transfected with FAT1-FAT2 gene)were 7.9%,7.03%,3.92%,5.64%,7.01%and 9.43%,and the results showed significant differences.Theω-6/ω-3 ratio decreased significantly from 1.4 in the control group to 0.42-1 in the experimental group.【Conclusion】FAT1-FAT2 mRNA can be expressed and produced by transfecting pig kidney cell PK15 with FAT1-FAT2 double gene by constructing target vector,and the content ofω-3 PUFAs in transfected cells can be significantly increased,which lay a foundation for the subsequent production of transfected pig with FAT1-FAT2 double gene.
作者
林纯
汤飞
李紫聪
LIN Chun;TANG Fei;LI Zicong(College of Nursing and Health,Lingnan Institute of Technology,Guangzhou 510663,China;South China Biosafety Level 4 Laboratory(Preparation),Sun Yat-sen University,Guangzhou 510275,China;College of Animal Science,South China Agricultural University/National Engineering Research Center for Breeding Swine Industry,Guangzhou 510642,China)
出处
《广东农业科学》
CAS
2021年第8期116-123,共8页
Guangdong Agricultural Sciences
