鸡Apob基因组织表达与CRISPR/Cas9敲除系统的构建

doi:10.11843/j.issn.0366-6964.2021.03.007

摘要/Abstract

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

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.

Key words: chicken, Apob gene, CRISPR/Cas9, lipid metabolism

中图分类号:

S831.2

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.

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.

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