携带 FAT1-FAT2 基因打靶载体构建及其在猪肾细胞的表达与功能鉴定

林　纯; 汤　飞; 李紫聪

文章摘要

林　纯，汤　飞，李紫聪.携带 FAT1-FAT2 基因打靶载体构建及其在猪肾细胞的表达与功能鉴定[J].广东农业科学,2021,48(8):116-123

查看全文 HTML 携带 FAT1-FAT2 基因打靶载体构建及其在猪肾细胞的表达与功能鉴定

Construction of FAT1-FAT2 Gene Targeting Vector and Identification of its Expression and Function in Porcine Kidney Cells

DOI：10.16768/j.issn.1004-874X.2021.08.014

中文关键词: 不饱和脂肪酸 FAT1-FAT2 基因打靶载体脂肪酸

英文关键词: polyunsaturated fatty acids FAT1-FAT2 gene targeting vector fatty acid

基金项目:

作者	单位
林　纯，汤　飞，李紫聪	1. 广东岭南职业技术学院护理与健康学院，广东广州 510663； 2. 中山大学华南生物安全四级实验室（筹），广东广州 510275； 3. 华南农业大学动物科学学院 / 国家生猪种业工程技术研究中心，广东广州 510642

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中文摘要:

【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。

英文摘要:

【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 modulated ω-3 polyunsaturated fatty acid dehydrogenase and Δ-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 of ω-6 PUFAs and ω-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. The ω-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 of ω-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.

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