| 鸡Apob基因组织表达与CRISPR/Cas9敲除系统的构建 |
| |
| 引用本文： | 吉琳,杨秋月,方斐旻,窦虹,郁建锋,徐璐,顾志良.鸡Apob基因组织表达与CRISPR/Cas9敲除系统的构建[J].畜牧兽医学报,2021,52(3):630-640. |
| |
| 作者姓名： | 吉琳 杨秋月 方斐旻 窦虹 郁建锋 徐璐 顾志良 |
| |
| 作者单位： | 常熟理工学院生物与食品工程学院, 苏州 215500 |
| |
| 基金项目： | 国家自然科学基金青年基金(31802050);江苏省自然科学基金(BK20191476)。 |
| |
| 摘 要： | 旨在探究Apob基因在鸡肝脂质代谢过程中的功能。本研究对鸡Apob蛋白进行理化性质分析；利用RT-qPCR检测Apob基因在4周龄黄羽肉鸡组织中的表达情况，每组设置3个重复，进行3次平行试验。根据鸡Apob蛋白关键结构域，在Apob基因外显子设计3对sgRNA，构建Cas/gRNA载体；将重组质粒转染DF-1细胞后，利用T7核酸内切酶I （T7 endonuclease I，T7EI）酶切法和TA克隆测序法筛选敲除活性位点并计算敲除效率。利用RT-qPCR检测基因敲除后亚克隆细胞中Apob基因mRNA表达情况。结果表明，鸡Apob的相对分子质量为523.356 ku，平均亲水性为-0.300，为稳定的蛋白质，并且该基因主要在鸡的肝、肾和小肠组织表达。敲除载体转染至DF-1细胞后，T7EI酶切发现，Cas/gRNA6、Cas/gRNA7和Cas/gRNA8三个位点均可发挥敲除活性，TA克隆测序结果表明，三者的敲除效率分别为33.3%、65%和80%。同时，RT-qPCR结果显示，转染Cas9/gRNA7、Cas9/gRNA6、Cas9/gRNA8的细胞中Apob基因mRNA表达水平约分别下调99.96%（P<0.01）、85%（P<0.01）、47%（P<0.05）。综上所述，本研究揭示了鸡Apob基因在组织中的表达特点和蛋白的理化性质；成功构建了鸡Apob基因CRISPR/Cas9敲除载体，并筛选出最佳敲除位点，获得了Apob基因敲除的亚克隆细胞，为进一步探索Apob基因在鸡肝中的功能奠定了基础。
|
| 关 键 词： | 鸡 Apob基因 CRISPR/Cas9 脂质代谢 |
| 收稿时间： | 2020-07-24 |
Tissue Expression and Construction of CRISPR/Cas9 Knockout System of Apob in Chicken |
| |
| JI Lin,YANG Qiuyue,FANG Feimin,DOU Hong,YU Jianfeng,XU Lu,GU Zhiliang.Tissue Expression and Construction of CRISPR/Cas9 Knockout System of Apob in Chicken[J].Acta Veterinaria et Zootechnica Sinica,2021,52(3):630-640. |
| |
| Authors: | JI Lin YANG Qiuyue FANG Feimin DOU Hong YU Jianfeng XU Lu GU Zhiliang |
| |
| Institution: | School of Biology and Food Engineering, Changshu Institute of Technology, Suzhou 215500, China |
| |
| Abstract: | The study aimed to explore the function of Apob gene in the lipid metabolism of chicken liver.The physical and chemical properties of chicken Apob protein were analyzed;RT-qPCR was used to detect the expression of Apob gene in the tissues of 4-week-old yellow broiler chickens,3 replicates were set up in each group and 3 parallel experiments were carried out.Three pairs of sgRNA were designed according to the sequence of key domains of chicken Apob protein to construct Cas/gRNA vectors;T7 endonuclease I(T7EI)digestion method and TA cloning sequencing method were used to screen knockout active sites and calculate gene knockout efficiency in cells after the Cas/gRNA vector was transfected into DF-1 cells;The mRNA expression of Apob gene was detected in subcloned cells using RT-qPCR after gene knockout.The results showed that the relative molecular mass of chicken Apob was 523.356 ku and the average hydrophilicity was-0.300,which was a stable protein.And the Apob gene mainly expressed in chicken liver,kidney and small intestine tissues.T7EI digestion result showed that Cas/gRNA vectors could knock out the gene effectively.TA clone sequencing results showed that knockout efficiency of 3 active sites(Cas/gRNA6,Cas/gRNA7 and Cas/gRNA8)were 33.3%,65%and 80%,respectively.At the same time,RT-qPCR results showed that the expression level of Apob genes in transfected Cas9/gRNA7,Cas9/gRNA6,Cas9/gRNA8 cells were down-regulated by about 99.96%(P<0.01),85%(P<0.01),47%(P<0.05),respectively.In summary,we revealed the expression characteristics of Apob gene in chicken tissues and the physicochemical properties of the protein;Then we contructed Cas/gRNA knockout vectors,screened the optimal knock sites,and established Apob knockout subclonal cells successfully,which laid a foundation for exploring the function of Apob gene in chicken liver. |
| |
| Keywords: | chicken Apob gene CRISPR/Cas9 lipid metabolism |
| 本文献已被 CNKI 维普 等数据库收录！ |
| 点击此处可从《畜牧兽医学报》浏览原始摘要信息 |
| 点击此处可从《畜牧兽医学报》下载免费的PDF全文 |
|
