Fat-1转基因克隆渤海黑牛研究

发布时间：2018-05-31 14:24

本文选题：Fat-1基因 + 体细胞克隆　；参考：《内蒙古大学》2015年硕士论文

【摘要】：Fat-1基因编码的产物是多不饱和脂肪酸脱氢酶,能将n-6多不饱和脂肪酸(PUFAs)转化为n-3 PUFAs。本研究利用MAR (matrix attachment regions)序列作为启动元件,构建了MAR-CAG-Fat-1-PolyA-MAR单顺反子表达盒,转染渤海黑牛胎儿成纤维细胞,通过有限稀释法挑取单克隆细胞进行培养,提取基因组DNA进行鉴定,获得了转基因阳性单克隆细胞系。然后,对转基因阳性细胞DNA整合,RNA、蛋白表达、脂肪酸含量等鉴定分析。用pMAR-CAG-Fat-1-PolyA-MAR载体(单酶切载体)和MAR-CAG-Fat-1-PolyA-MAR载体(双酶切载体)分别制备转基因小鼠。将表达良好的转基因单克隆细胞通过体细胞核移植技术制备克隆胚胎。结果表明,(1)获得了稳定转染双酶切载体的单克隆阳性细胞系,该细胞系的n-3 PUFAs表达水平升高且n-6/n-3PUFAs的比值降低。脂肪酸合成基因FASN、ACC、SREBP-1表达与对照相比明显降低,脂肪酸代谢基因PPARA、PPARG降低。细胞中fat1蛋白表达。(2)获得了双酶切和单酶切载体的转基因小鼠。其中转基因双酶切鼠FO代7只,F1代3只,F2代2只；转基因单酶切鼠F0代7只,F1代28只。转基因双酶切鼠F0、F1和F2代鼠的肌肉和脂肪组织中n-3 PUFAs含量均升高,n-6/n-3PUFAs比值均降低；转基因单酶切鼠肌肉和脂肪组织中n-3 PUFAs含量降低,n-6/n-3 PUFAs比值升高。(3)双酶切鼠肌肉组织中脂肪酸相关基因实时定量分析,发现脂肪酸合成基因FASN、ACC、SREBP-1表达与对照相比明显降低,脂肪酸代谢基因PPARA、PPARG表达降低。(4)蛋白差异表达和动态定量分析,发现单酶切组试验差异蛋白有99个,其中上调蛋白62个,下调蛋白37个；双酶切组试验差异蛋白有118个,其中上调蛋白81个,下调蛋白37个。(5)利用表达良好的转基因细胞系为核供体生产24枚克隆胚胎,移植受体母畜12头,有2头妊娠。其中1头发育到期并生下健康犊牛。上述结果表明,MAR-CAG-Fat-1-PolyA-MAR载体的转基因细胞、转基因小鼠均有良好表达,而且可以生产转基因克隆牛。
[Abstract]:The product encoded by Fat-1 gene is polyunsaturated fatty acid dehydrogenase, which can transform n-6 polyunsaturated fatty acid (PUFas) into n-3 PUFAs. In this study, the MAR matrix attachment regions sequence was used as the starting element to construct the MAR-CAG-Fat-1-PolyA-MAR monocistron expression box, which was transfected into Bovine fetal fibroblasts in Bohai Sea. The monoclonal cells were cultured by limited dilution method, and genomic DNA was extracted for identification. Transgenic monoclonal cell lines were obtained. Then, DNA integration RNAs, protein expression and fatty acid content of transgenic positive cells were identified and analyzed. Transgenic mice were prepared by pMAR-CAG-Fat-1-PolyA-MAR vector (single enzyme digestion vector) and MAR-CAG-Fat-1-PolyA-MAR vector (double enzyme digestion vector). The cloned embryos were prepared by somatic cell nuclear transfer. The results showed that Monoclonal positive cell lines stably transfected with double enzyme digestion vector were obtained. The expression of n-3 PUFAs increased and the ratio of n-6/n-3PUFAs decreased. The expression of fatty acid synthase gene ACCS SREBP-1 was significantly lower than that of control, and the fatty acid metabolism gene PPARAA PPARG was decreased. Transgenic mice with double enzyme digestion and single enzyme digestion vector were obtained. Among them, there were 2 mice in F _ 2 generation and 7 F _ 1 generation in FO generation and 28 F _ 1 generation in F _ 0 generation of transgenic mice. The content of n-3 PUFAs in muscle and adipose tissue of F0 / F 1 and F 2 generation mice was increased and the ratio of n-6 / n-3 PUFAs was decreased. The content of n-3 PUFAs in muscle and adipose tissue of transgenic mice was decreased. The ratio of n-6 / n-3 PUFAs was increased. The results of real-time quantitative analysis of fatty acid related genes in muscle tissue of mice by double enzyme digestion showed that the expression of fatty acid synthase gene FASNN-ACC-SREBP-1 was significantly lower than that of control. The differential expression and dynamic quantitative analysis of fatty acid metabolism gene PPARA-PPARG) showed that there were 99 differentially expressed proteins in single-enzyme digestion group, including 62 up-regulated proteins, 37 down-regulated proteins, and 118 differentially expressed proteins in double-enzyme digested group. Among them, 81 upregulated proteins and 37 down-regulated proteins were used to produce 24 cloned embryos from well-expressed transgenic cell lines. One of the calves matured and gave birth to healthy calves. The results showed that the transgenic cells of MAR-CAG-Fat-1-PolyA-MAR vector were well expressed in transgenic mice and could produce transgenic cloned cattle.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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