本文主要研究内容
作者广璐,张英,郭晶,白春玲,魏著英,于超然,扈廷茂,李光鹏（2019）在《CRISPR/Cas9介导h FAD3基因在牛NCAPG-LCORL位点的定点整合》一文中研究指出：亚麻(Linum usitatissimum)脂肪酸去饱和酶基因3(fatty acid desaturase 3, FAD3)是一种脂肪酸脱氢酶基因,编码ω-3多不饱和脂肪酸(polyunsaturated fatty acids, PUFAs)脱氢酶,能够将ω-6 PUFAs转化成ω-3PUFAs。本研究利用CRISPR/Cas9介导,将无抗性、无标记的人(Homo sapiens)源化h FAD3基因表达载体C2(5’同源臂-CAG-hFAD3-PolyA-3’同源臂)和M2(5’同源臂-MAR-CAG-hFAD3-PolyA-MAR-3’同源臂)定点敲入牛(Bos taurus)胎儿成纤维细胞NCAPG-LCORL位点。运用气质联用仪对脂肪酸含量进行分析,结果表明hFAD3转基因细胞中ω-6/ω-3 PUFAs比值(0.565)较非转基因细胞(1.549)显著下降(P<0.01)。qRT-PCR结果表明h FAD3基因整合并表达后,脂肪分解相关的过氧化物酶体增殖物激活受体基因(peroxisome proliferator activated receptor1, PPARg)、激素敏感脂肪酶基因(hormone-sensitive lipase, LIPE)和脂蛋白脂酶(lipoprotein lipase, LPL)表达总量上调倍数大于脂肪合成相关的乙酰辅酶A羧化酶基因(acetyl CoA carboxylase, ACC)、硬脂酰辅酶A去饱和酶基因(stearoyl CoA desaturase, SCD)和脂肪酸合酶基因(fatty acid synthase, FASN)表达总量,说明hFAD3基因的表达使得细胞内的脂肪趋于分解。通过流式分选获得59株C2打靶载体单克隆细胞株,PCR鉴定及测序结果表明,其中定点整合阳性单克隆细胞3株,定点整合效率为5.1%。同时检测定点整合阳性单克隆细胞株中脂肪酸的含量以及脂肪相关基因的表达量,结果与转染后细胞一致。比较有无核基质结合区(matrix attachment region, MAR)序列介导两种转基因细胞中hFAD3基因的表达量,结果显示,MAR介导组是无MAR组的5.91倍(P<0.01)。本研究利用CRISPR/Cas9介导可实现hFAD3基因在牛NCAPG-LCORL位点的定点整合以及在细胞水平正常行使其生物学功能,同时MAR的介导可显著提高外源基因的表达量,为安全高效生产转基因动物提供科学依据。
Abstract
ya ma (Linum usitatissimum)zhi fang suan qu bao he mei ji yin 3(fatty acid desaturase 3, FAD3)shi yi chong zhi fang suan tuo qing mei ji yin ,bian ma ω-3duo bu bao he zhi fang suan (polyunsaturated fatty acids, PUFAs)tuo qing mei ,neng gou jiang ω-6 PUFAszhuai hua cheng ω-3PUFAs。ben yan jiu li yong CRISPR/Cas9jie dao ,jiang mo kang xing 、mo biao ji de ren (Homo sapiens)yuan hua h FAD3ji yin biao da zai ti C2(5’tong yuan bei -CAG-hFAD3-PolyA-3’tong yuan bei )he M2(5’tong yuan bei -MAR-CAG-hFAD3-PolyA-MAR-3’tong yuan bei )ding dian qiao ru niu (Bos taurus)tai er cheng qian wei xi bao NCAPG-LCORLwei dian 。yun yong qi zhi lian yong yi dui zhi fang suan han liang jin hang fen xi ,jie guo biao ming hFAD3zhuai ji yin xi bao zhong ω-6/ω-3 PUFAsbi zhi (0.565)jiao fei zhuai ji yin xi bao (1.549)xian zhe xia jiang (P<0.01)。qRT-PCRjie guo biao ming h FAD3ji yin zheng ge bing biao da hou ,zhi fang fen jie xiang guan de guo yang hua wu mei ti zeng shi wu ji huo shou ti ji yin (peroxisome proliferator activated receptor1, PPARg)、ji su min gan zhi fang mei ji yin (hormone-sensitive lipase, LIPE)he zhi dan bai zhi mei (lipoprotein lipase, LPL)biao da zong liang shang diao bei shu da yu zhi fang ge cheng xiang guan de yi xian fu mei Asuo hua mei ji yin (acetyl CoA carboxylase, ACC)、ying zhi xian fu mei Aqu bao he mei ji yin (stearoyl CoA desaturase, SCD)he zhi fang suan ge mei ji yin (fatty acid synthase, FASN)biao da zong liang ,shui ming hFAD3ji yin de biao da shi de xi bao nei de zhi fang qu yu fen jie 。tong guo liu shi fen shua huo de 59zhu C2da ba zai ti chan ke long xi bao zhu ,PCRjian ding ji ce xu jie guo biao ming ,ji zhong ding dian zheng ge yang xing chan ke long xi bao 3zhu ,ding dian zheng ge xiao lv wei 5.1%。tong shi jian ce ding dian zheng ge yang xing chan ke long xi bao zhu zhong zhi fang suan de han liang yi ji zhi fang xiang guan ji yin de biao da liang ,jie guo yu zhuai ran hou xi bao yi zhi 。bi jiao you mo he ji zhi jie ge ou (matrix attachment region, MAR)xu lie jie dao liang chong zhuai ji yin xi bao zhong hFAD3ji yin de biao da liang ,jie guo xian shi ,MARjie dao zu shi mo MARzu de 5.91bei (P<0.01)。ben yan jiu li yong CRISPR/Cas9jie dao ke shi xian hFAD3ji yin zai niu NCAPG-LCORLwei dian de ding dian zheng ge yi ji zai xi bao shui ping zheng chang hang shi ji sheng wu xue gong neng ,tong shi MARde jie dao ke xian zhe di gao wai yuan ji yin de biao da liang ,wei an quan gao xiao sheng chan zhuai ji yin dong wu di gong ke xue yi ju 。
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论文作者分别是来自农业生物技术学报的广璐,张英,郭晶,白春玲,魏著英,于超然,扈廷茂,李光鹏，发表于刊物农业生物技术学报2019年01期论文，是一篇关于脂肪酸去饱和酶基因论文,定点整合论文,核基质结合区论文,农业生物技术学报2019年01期论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自农业生物技术学报2019年01期论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。